Degradation of 1,4-Dichlorobenzene by Alcaligenes sp. Strain ... - NCBI

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Jul 7, 1986 - They have been detected in many surface waters and ... sp. from an enrichment culture with 1,4-DCB provided as the sole ... 7H20, 1 g of NH4NO3, 50mg of ... above-mentioned trace-element solution (29), and 0.1 ml ofa .... ride atoms) was measured after addition of 0.7 ml of cell ..... Chemical structure and.

Vol. 52, No. 6

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1986, p. 1374-1381

0099-2240/86/121374-08$02.00/0 Copyright © 1986, American Society for Microbiology

Degradation of 1,4-Dichlorobenzene by Alcaligenes sp. Strain A175 GOSSE SCHRAA,* MARTIN L. BOONE, MIKE S. M. JETTEN, ALEX R. W. VAN NEERVEN, PATRICIA J. COLBERG,t AND ALEXANDER J. B. ZEHNDER Department of Microbiology, Agricultural University, 6703 CT Wageningen, The Netherlands Received 7 July 1986/Accepted 17 September 1986

An organism, identified as an Alcaligenes sp., was isolated from an enrichment culture in which 1,4-dichlorobenzene served as the sole carbon and energy source. During growth with 1,4-dichlorobenzene in pure culture, stoichiometric amounts of chloride were released. Growth experiments and oxygen uptake rates with other chlorinated aromatic compounds revealed a high degree of specificity of the initial dioxygenase. cis-1,2-Dihydroxycyclohexa-3,5-diene oxidoreductase and 1,2-pyrocatechase, but not 2,3-pyrocatechase, were found in cell extracts, while 3,6-dichlorocatechol and (2,5-dichloro)muconic acid could be detected as intermediates during degradation of 1,4-dichlorobenzene. It is proposed that dioxygenases are involved in the initial steps of 1,4-dichlorobenzene degradation, while ring opening proceeds via ortho cleavage.

MATERIALS AND METHODS

The extensive use over the past few decades of chlorinated benzenes as organic solvents, insecticides, and odorizers and their production as intermediates during the synthesis of chemicals has led to a wide distribution of these highly volatile, lipophilic compounds in the environment. They have been detected in many surface waters and groundwaters, in sewage, and in some biological tissues (22). The observed levels are, in general, too low to cause immediate acute toxicity to mammals, birds, and aquatic organisms (22), but little information exists about long-term exposure and bioaccumulation of chlorinated benzenes. Since chlorinated benzenes are chemically stable in nature and since photochemical degradation does not play an important role in soil and aquatic environments (27), breakdown via enzymatically catalyzed reactions is the only way these compounds are eliminated from these environments. Little information is available about the biodegradation of dichlorobenzenes. Oxidation of all three isomers of dichlorobenzene to the respective chlorinated catechols by Pseudomonas putida was shown by Kilbourn and Gibson (R. G. Kilbourn and D. T. Gibson, Abstr. Annu. Meet. Am. Soc. Microbiol. 1979, N70, p. 191). de Bont et al. (6) described a microorganism that grows on 1,3-dichlorobenzene (1,3-DCB). A dioxygenase-mediated reaction resulted in 3,5-dichlorocatechol, which subsequently underwent ortho cleavage to yield C02, HCI, and H20. Dechlorination did not occur until after the ring opening. 1,4-DCB degradation in laboratory aquifer columns and in field situations has been observed by Kuhn et al. (18). Schraa et al. (24) and Schwarzenbach et al. (25) reported that, in addition to degradation in laboratory aquifer columns, growth of bacteria occurs in batch cultures with 1,4-DCB as the sole carbon and energy source. Recently, bacteria have been isolated which are able to completely mineralize 1,4-DCB (W. Reineke, personal communication). In this paper, we describe the isolation of an Alcaligenes sp. from an enrichment culture with 1,4-DCB provided as the sole carbon and energy source, its metabolic properties, and the proposed steps in the initial degradation pathway.

Enrichment and isolation. Strain A175 was isolated from an inoculum obtained from a mixture of various soil and water samples. For the enrichment, we used 100-ml serum bottles to which 40 ml of mineral medium was added. 1,4-DCB served as the sole carbon and energy source and was supplied in the gaseous phase as follows. A small reagent tube was placed into the serum bottle with its open end well above the medium level. Some crystals of the chemical were added to the tube. The bottles were sealed with a viton septum (Iso-versinic, Rubber B.V., Hilversum, Netherlands). Because of its volatility (vapor pressure, 0.6 mm at 20°C), 1,4-DCB evaporated into the headspace and subsequently dissolved in the medium. The mineral salts medium consisted of (per liter of demineralized water) 1.4 g of Na2HPO4. 2H20, 0.7 g of KH2PO4, 0.3 g of NH4Cl, 0.1 g of MgSO4 7H20, 10 mg of CaCl2. 2H20, 1 g of NaNO3, 2.5 mg of yeast extract, and 1 ml of a trace-element solution (29). The bottles were incubated at 30°C. The headspace served as the source of oxygen. Upon growth, subcultures were made and serial dilutions were plated on nutrient agar and incubated at 30°C. Several colonies were isolated from the plates and transferred to serum bottles as described above. This time, 1,4-DCB crystals were added directly to a mineral salts medium, low in chloride, which consisted of (per liter of demineralized water) 1.4 g of Na2HPO4. 2H20, 0.7 g of KH2PO4, 0.1 g of MgSO4. 7H20, 1 g of NH4NO3, 50 mg of Ca(NO3)2 4H20, 2.5 mg of yeast extract, 1 ml of the above-mentioned trace-element solution (29), and 0.1 ml of a vitamin solution (the solubility of 1,4-DCB in water at 25°C is 79 mg/liter). The vitamin solution contained (per liter of demineralized water) 2 mg of biotin, 2 mg of folic acid, 10 mg of pyridoxal hydrochloride, 5 mg of riboflavin, 5 mg of thiamine, 5 mg of nicotinic acid, 5 mg of calcium pantothenate, 5 mg of cyanocobalamin, and 5 mg of p-aminobenzoic acid. After incubation, two different organisms were isolated, of which one, designated as strain A175, was studied in detail. During the study, this strain was maintained on nutrient agar slants. Growth conditions. Cells were usually grown in 100-ml serum bottles as described above. Mass cultures were grown in 5-liter Erlenmeyer flasks, each containing 1 liter of medium and sealed with a rubber stopper. 1,4-DCB was added in the gaseous phase as follows. A reagent tube with an

* Corresponding author. t Present address: Swiss Federal Institute for Water Resources and Water Pollution Control, CH-8600 Dubendorf, Switzerland.

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VOL. 52, 1986

opening on the side about 3 cm from the bottom was inserted into the rubber stopper in such a way that the open top of the tube reached above the top of the stopper and the side opening was still below the stopper. 1,4-DCB could thus be aseptically added to the tube, which after receiving the substrate was sealed with a small stopper. Through the side opening, the substrate could evaporate into the headspace, which also served as the oxygen reservoir. Bottles and flasks were incubated at 30°C on a rotary shaker (63 rpm). Preparation of washed suspensions of bacteria and cell extracts. Organisms were harvested from the 5-liter Erlenmeyer flasks by centrifugation (20,000 x g for 20 min at 4°C), washed twice in 50 mM phosphate buffer (pH 7.0), and resuspended in the same buffer. Before centrifugation, organisms were checked for purity by being plated on nutrient agar and by microscopic inspection. For preparation of cell extracts, washed cell suspensions were subjected to ultrasonic disintegration (10 20-s bursts at 0°C) followed by centrifugation at 30,000 x g for 20 min at 4°C. The clear supernatant was used as the crude cell extract. Protein concentrations were measured by the method of Lowry et al. (19). Analytical methods. Concentrations of 1,4-DCB were determined by capillary gas chromatography after extraction of incubation mixtures with hexane containing 1,3,5trichlorobenzene as an internal standard. After addition of 6 ml of hexane and vigorous shaking of the incubation mixtures for 1 min, 1 ,ul of hexane extract was analyzed with a Kipp Analytica 8200 gas chromatograph. The unit was equipped with a flame ionization detector fitted to a fused silica column (CP Wax 57 CB, 10 m by 0.33 mm [inner diameter]) which was kept at 80°C. Chloride ion concentrations were determined with a Marius Micro-chlor-o-counter (Marius, Utrecht, Netherlands) with an NaCl solution as the standard. Metabolites, produced from either washed-cell suspensions or cell extracts, were analyzed by reverse-phase highpressure liquid chromatography (LKB 2150 pump and 2152 controller; Ultra Pac column [250 by 4 mm] filled with Lichrosorb RP-8 and RP-18 [diameter, 10 ,um] preceded by a universal Chrompack RP column [75 by 2.1 mm]). The mobile phase was acetonitrile-water (ratio by volume, 60:40), the flow rate was 1 ml/min, and 20- and 50-pul samples were analyzed with a Uvicord SD detector at 206 and 280 nm and quantified with a Spectra Physics 4270 computing integrator. Metabolites were identified by comparison of their retention times with those of pure substances and by scanning UV spectra. Growth was determined by direct microscopic counting with a Burker Turk counting chamber and by measuring the protein concentration by the method of Lowry et al. (19) with bovine serum albumin as the standard. In the samples which were also analyzed for 1,4-DCB, the hexane was allowed to evaporate before protein analysis. Bacterial DNA (moles percent guanine plus cytosine) was determined by the method described by de Bont et al. (5). Measurement of oxygen uptake. Oxygen uptake rates were measured polarographically with an oxygen electrode at 30°C. Initially, the endogeneous oxygen uptake of 0.1 ml of freshly washed cells or cell extracts in 2.8 ml of 50 mM phosphate buffer (pH 7.0) or 40 mM Tris hydrochloride buffer (pH 8.0) was measured for 4 min. The reaction was started by injecting 0.1 ml of the assay substrate, and oxygen uptake was monitored for 5 min. Substrates were generally added as saturated solutions. Catechol and cis-1,2dihydroxycyclohexa-3,5-diene were added as 10 mM solu-

1,4-DICHLOROBENZENE DEGRADATION

1375

tions, while acetate, butyrate, pyruvate, succinate, and glucose were added as 1-g/liter solutions. Oxygen uptake rates were corrected for endogenous consumption in both the cell suspension and the cell extract. Enzyme assays. All enzyme assays were performed at 30°C

with a Beckman spectrophotometer (Beckman Instruments, Inc., Fullerton, Calif.) or polarographically with an oxygen electrode. Dichlorobenzene dioxygenase, assumed to catalyze the initial step in the degradation pathway (15), was measured either by determining NAD(P)H oxidation at 340 nm or by following oxygen uptake. Reaction mixtures contained (in 3 ml) 2.6 ml of 25 mM phosphate buffer (pH 7.5), 0.1 ml of 1 mM FeSO4 7H20, 0.1 ml of 0.33 mM NAD(P)H, 0.1 ml of the respective substrate, and 0.1 ml of cell extract (0.1 to 0.2 mg of protein). The dioxygenase activity was also determined with cell extracts which contained either 10% acetone or 10% ethanol as antioxidants. cis-1,2-Dihydroxycyclohexa-3,5-diene oxidoreductase was measured by determining NAD+ reduction at 340 nm (23). Reaction mixtures contained (in 3 ml) 2.7 ml of 50 mM phosphate buffer (pH 7.5), 0.1 ml of 20 mM NAD+, 0.1 ml of 10 mM cyclohexadienediol, and 0.1 ml of cell extract (0.1 to 0.2 mg of protein).

1,2-Pyrocatechase (catechol 1,2-dioxygenase)

was mea-

2,3-Pyrocatechase (catechol 2,3-dioxygenase)

was mea-

sured by determining the formation of (substituted) muconic acid at 260 nm (7). Reaction mixtures contained (in 3 ml) 2.7 ml of 40 mM Tris hydrochloride buffer (pH 8.0), 0.1 ml of 40 mM EDTA, 0.1 ml of 10 mM (substituted) catechol, and 0.1 ml of cell extract (0.1 to 0.2 mg of protein).

sured by determining the formation of muconic acid semialdehyde at 375 nm (21). Reaction mixtures contained (in 3 ml) 2.7 ml of 50 mM phosphate buffer (pH 7.5), 0.1 ml of 1 mM FeSO4 7H20 or 1 mM FeC13, 0.1 ml of 10 mM catechol, and 0.1 ml of cell extract (0.1 to 0.2 mg of protein) to which 10% acetone or 10% ethanol had been added. The activity in cell extract which had undergone heat treatment at 55°C for 10 min was also determined. Metabolites. Incubation experiments for metabolite determination were performed at 30°C. 3,6-Dichlorocatechol was measured after addition of 10 ml of a washed-cell suspension (30 mg of protein) to 90 ml of mineral salts medium containing 68 pumol of 1,4-DCB per liter. 2,5-Dichloromuconic acid (possibly with one or two chloride atoms) was measured after addition of 0.7 ml of cell extract (1.7 mg of protein) and 0.3 ml of 20 mM 3,6dichlorocatechol to 9.0 ml of 40 mM Tris hydrochloride buffer (pH 8.0). 2,5-Dimethylmuconic acid was measured after addition of 1.0 ml of cell extract, 0.3 ml of 40 mM EDTA, and 0.3 ml of 33 mM 3,6-dimethylcatechol to 8.4 ml of 40 mM Tris hydrochloride buffer (pH 8.0). Chemicals. 3-Chlorocatechol was synthesized by the conversion of 2-chlorophenol to 3-chloro-2-hydroxybenzaldehyde by the method of Duff (8) or Reimer and Tiemann (28) and by the subsequent oxidation of the benzaldehyde through the Dakin (4) or Baker et al. (1) reaction. The same methods were used to synthesize 3-methylcatechol from 2-hydroxytoluene, 3,6-dichlorocatechol from 2,5dichlorophenol, 3,6-dimethylcatechol from 2,5dimethylphenol, and 4-chlorocatechol from 4-chlorophenol. Benzene, chlorobenzene, 1,2-, 1,3-, and 1,4dichlorobenzenes, 1,2,3- and 1,2,4-trichlorobenzenes, phenol, 2-, 3-, and 4-chlorophenols, 2,3-, 2,4-, 2,5-, 3,4-, and 3,5-dichlorophenols, 2,3,5- and 2,4,5-trichlorophenols, toluene, 1,2-, 1,3-, and 1,4-dimethylbenzenes, 2-, 3-, and 4-

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FIG. 1. Cells of Alcaligenes sp. strain A175 grown on 1,4-dichlorobenzene. (A) Phase-contrast micrograph; bar represents 5 ,um; (B) scanning electron micrograph, showing flagellar arrangement, negatively stained; bar represents 0.5 ,um; (C) transmission electron micrograph, showing a cross-section of a cell; bar represents 0.2 ,um.

hydroxytoluenes, 2,5-dimethylphenol, benzoate, 4hydroxybenzoate, 4-chlorobenzoate, and catechol were purchased from Janssen Chimica, Beerse, Belgium. cis-1,2Dihydroxycyclohexa-3,5-diene was a generous gift from Will van den Tweel. Biochemicals were obtained from Boehringer GmbH, Mannheim, Federal Republic of Germany. All other chemicals were of analytical grade and were used without further purification. RESULTS Isolation and characterization of the pure culture. With the above-described enrichment method, it took about 2 months before degradation of 1,4-DCB was observed. Initially, no isolates could be obtained from the serial dilutions on nutrient agar plates. However, after about 10 months, during which numerous subcultures were made, two different bacteria growing on 1,4-DCB were isolated from tiny, cream colonies. The long period required for isolation was observed previously with bacteria exposed to xenobiotic compounds (17, 20, 23). The two organisms subsequently lost their ability to grow on 1,4-DCB after about five successive transfers on a nonselective medium containing no 1,4-DCB. One of the two isolated bacteria, strain A175, was a strictly aerobic, gram-negative, motile, rod-shaped organism with peritrichous flagella (Fig. 1). The guanine-plus-cytosine content of the DNA was found to be 75.8 mol%. The strain is catalase and oxidase positive and shows a positive reaction in the Hugh-Leifson test (14). No denitrification is observed during growth on acetate. The organism is able to grow on acetate, propionate, butyrate, DL-,3-hydroxybutyrate, succinate, glucose, and xylose but not on pyruvate, lactate, 1-malate, 2-oxoglutarate, fumarate, malonate, ethanol, methanol, methylamine hydrochloride, or hexane. On the basis of the above characteristics, the organism was tentatively classified as an Alcaligenes species (13). Growth of strain A175 with 1,4-DCB. The ability of strain A175 to grow with 1,4-DCB as a sole source of carbon and energy is shown in Fig. 2. Growth, expressed as protein increase, was associated with the release of chloride ions. The doubling time of the organism during the exponential growth phase in batch culture was about 8 h. Incubation of strain A175 with increasing amounts of 1,4-DCB resulted in a linear relationship between the amount of dichlorobenzene added and the increase in cell

number and protein (Fig. 3). The experiment was done in seven separate serum bottles in which 1,4-DCB crystals were added directly to the medium. The incubation lasted for 4 days, after which 1,4-DCB could no longer be detected. The stoichiometric amounts of chloride which were measured after consumption of 1,4-DCB and the increase in protein strongly suggest a complete mineralization of the added substrate. # The effect of temperature on growth was tested by incubation of the organism for 2 days at seven different temperatures. Maximum growth and maximum chloride release occurred at around 29°C (Fig. 4). Growth with other aromatic substrates. Apart from the previously mentioned substrates, growth of strain A175 was tested on a number of additional compounds (Table 1). Most of these compounds were chosen because of their relevance in the elucidation of the 1,4-DCB degradation pathway. The results suggest that the substrate range of strain A175 is very limited. Owing to the absence of growth on the phenolic compounds, it may be tentatively concluded that (i) no mono-oxygenases are involved in the initial steps in the

100 6

w

Li)(as as

4 o cu 0 X_

0

CL

3 2 time (days) FIG. 2. Protein concentration and chloride release after growth of Alcaligenes sp. strain A175 on 1,4-DCB in sealed 100-ml serum bottles with 40 ml of mineral salts medium (low in chloride). 1,4-DCB was supplied in the gaseous phase by addition of some crystals of the chemical to a small reagent tube in the bottle.

VOL. 52, 1986

1,4-DICHLOROBENZENE DEGRADATION 7

12

60

6

6

-

bacteria-6 lO

dtpz4 6rti 1 2 3

60 -

(U

TABLE 1. Growth of Alcaligenes sp. strain A175 on various carbon sourcesa Growth Compound Growth Compound

E

-0

0

.0~~~~~~~~~~~~ 40 -ro n4J 30

~~~~~~~~~~~3

42 -7 CJ~~~~~~~~1 lF - ihlrbnzn 20-H 2 C nraei n co.3 ,-C osmtinvru protein, 10 r6 cras in ehnubrtolarifchoie,addecesinpafr 0 0 0 0 123

1,4-dichtorobenzene (mM) FIG. 3. 1,4-DCB consumption versus increase in protein, innumber, release of chloride, and decrease in pH after growth of Alcaligenes sp. strain A175 with various concentrations of 1,4-DCB. 1,4-DCB crystals were added directly to 40 ml of mineral salts medium (low in chloride) in sealed 100-ml serum bottles. Analyses were performed after total removal of 1,4-DCB. Concentrations of 1,4-DCB were calculated as if the substrate were totally dissolved in the medium. crease in cell

metabolic pathway and (ii) degradation does not start with a dechlorination of 1,4-DCB to 4-chlorophenol. Oxygen uptake by whole cells and cell extracts. The oxygen uptake of washed cells and cell extracts of strain A175 pregrown on monochlorobenzene, 1,3-DCB, 1,4-DCB, benzene, and toluene was tested with a number of compounds (Tables 2 and 3). For the phenolic compounds in Table 1, no oxygen uptake could be detected. cis-1,2-Dihydroxycyclohexa-3,5-diene and the catechols, however, showed high oxygen uptake rates. The complete absence of oxygen uptake with the chlorinated benzenes by cell extracts points to the instability of the initial dioxygenase. This is in contrast to the uptake rates with catechols. Cells were unable to

50--4 E E

40

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roteincocnrtoanchoierlaefergwh

0

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40 35 30 incubation temperature

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FIG. 4. Protein concentration and chloride release after growth of Alcaligenes sp. strain A175 at different incubation temperatures. Growth took place in 100-ml serum bottles with mineral salts medium (low in chloride) and with an excess of 1,4-DCB. Incubation time was 48 h.

Benzene Monochlorobenzene 1,2-DCB 1,3-DCB 1,4-DCB 1,2,4-Trichlorobenzene

Phenol 2-Chlorophenol 3-Chlorophenol 4-Chlorophenol 2,3-Dichlorophenol 2,5-Dichlorophenol 3,4-Dichlorophenol 3,5-Dichlorophenol

+ +

+ +

-

-

-

-

2,4,5-Trichlorophenol Pentachlorophenol Catechol Toluene 1,2-Dimethylbenzene 1,3-Dimethylbenzene 1,4-Dimethylbenzene 2-Hydroxytoluene 3-Hydroxytoluene 4-Hydroxytoluene Benzoate 4-Hydroxybenzoate 4-Chlorobenzoate

-

+

+b

+b

2,4-Dihydroxybenzoate a Alcaligenes sp. strain A175 was grown in 100-ml serum bottles with mineral salts medium. Each compound was added as the sole carbon source in the gaseous phase. Growth was judged by increase in turbidity after 2. 4, and 10 days of incubation. b Growth occurred only when organisms were pregrown on benzene.

oxidize benzoate, toluene, and the three isomers of xylene and cresol, while the oxygen uptake rates with acetate, butyrate, pyruvate, and succinate were very low or undetectable. Experiments with the nonchlorinated compounds benzene, toluene, and acetate demonstrated the inducibility of the initial enzyme responsible for 1,4-DCB degradation. Oxygen uptake rates determined for the chlorinated benzenes by cells which were pregrown on benzene and toluene were very much reduced, while cells pregrown on acetate did not show any detectable oxygen uptake activity with any of the tested compounds except acetate and butyrate (data not shown). Enzyme activities in cell extracts. We were unable to detect any dichlorobenzene dioxygenase activity either by determining NAD(P)H oxidation at 340 nm or by following oxygen uptake. The enzyme activities of cis-1,2dihydroxycyclohexa-3,5-diene oxidoreductase and 1,2pyrocatechase were induced in cells of strain A175 when grown on benzene, toluene, monochlorobenzene, 1,3dichlorobenzene, or 1,4-dichlorobenzene (Table 4). However, no 2,3-pyrocatechase activity could be detected. The 1,2-pyrocatechase showed a higher activity for catechols with substituents on the 3- and 6-position than on the 4-position, while no activity was observed with 3,4dimethylcatechol and 3,4-dihydroxyphenylacetic acid (results not shown) as assay substrates. A consistently higher activity of oxidoreductase compared with that of 1,2pyrocatechase was observed. Metabolites. Incubation of a concentrated washed-cell suspension in mineral salts medium with 68 ,umol of 1,4-DCB per liter resulted in the temporary formation of a metabolite with a maximum concentration of 38 ,umol/liter after about 2

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SCHRAA ET AL.

TABLE 2. Relative oxygen uptake rates with various compounds by washed-cell suspensions of Alcaligenes sp. strain A1751 % Oxygen uptake for growth substrate:

Assay substrate

1,3-DCB 229 353 67 100 (74) 143 14 0 76 269 201 86 150 386 117 0 0 0 0 43 29 0 0

Monochlorobenzene

62 100 (144) 0 35 80 10 0 NDd 114 114 22 125 130 130 0 0 0 0 14 16 20 0

Benzene Monochlorobenzene 1,2-Dichlorobenzene 1,3-Dichlorobenzene 1,4-Dichlorobenzene 1,2,4-Trichlorobenzene Phenolsb CDHCHDC Catechol 3-Chlorocatechol 4-Chlorocatechol 3,6-Dichlorocatechol 3-Methylcatechol 3,6-Dimethylcatechol Toluene 1,2-, 1,3-, and 1,4-Dimethylbenzenes 2-, 3-, and 4-Hydroxytoluenes Benzoate Acetate Butyrate Pyruvate Succinate

1,4-DCB 122 132 27 52 100 (110) 10 0 140 177 112 86 128 125 143 0 0 0 0 20 20 0 0

Benzene

Toluene

100 (110) 45 0 0 40 0 0 125 105 125 45 150 143 126 0 0 0 0 0 ND ND ND

100 (85) 80 0 40 80 0 0 ND 127 190 18 82 127 120 0 0 0 0 0 ND ND ND

a Oxygen uptake rates are expressed as percentages of those for the individual growth substrates taken as 100%. For toluene-grown cells, benzene was the reference compound (no oxygen uptake was observed with toluene as an assay substrate). Absolute activities (as nanomoles of 02 per minute per milligram of protein) are given in parentheses for the relative rates reported as 100%. b The same phenols were tested as those listed in Table 1. c CDHCHD, cis-1,2-Dihydroxycyclohexa-3,5-diene. d ND, Not determined.

h. This compound, which was identified as 3,6-dichlorocatechol, could no longer be detected after 3 h. An additional metabolite was observed in an experiment in which cell extract was incubated with 3,6-dichlorocatechol in a Tris hydrochloride buffer. The intermediate, which reached its maximum concentration after 45 min, had a UV spectrum that showed a strong resemblance to the spectrum of (2,5dichloro)muconic acid (12). Incubation of cell extract (which exhibited 1,2- but not 2,3-pyrocatechase activity) with 3,6-dimethylcatechol in a Tris hydrochloride buffer with EDTA resulted in a rapid decline in the catechol concentration (Fig. 5). Simulta-

neously, 2,5-dimethylmuconic acid could be detected as a product (high-pressure liquid chromatography and UV spectrum scanning) but only up to a concentration of 0.2 mM. After 30 min, the medium started to become red. DISCUSSION The initial inability to isolate an organism able to grow on 1,4-DCB from a mixed culture in which degradation of 1,4-DCB took place is an indication of the absence of complete genetic information in one organism required to mineralize this xenobiotic compound. The ultimate isolation, after 10 months, of two different organisms, each capable of

TABLE 3. Relative oxygen uptake rates with various compounds by cell extracts of Alcaligenes sp. strain A175' % Oxygen uptake for growth substrate:

Assay substrate

Benzene Monochlorobenzene

1,2-DCB 1,3-DCB 1,4-DCB 1,2,4-Trichlorobenzene Phenols" CDHCHDc Catechol 3-Chlorocatechol 4-Chlorocatechol

3,6-Dichlorocatechol 3-Methylcatechol

3,6-Dimethylcatechol

Monochlorobenzene

0 0 0 0 0 0 0

NDd 100 (305) 100 21 51 43 114

1,3-DCB

1,4-DCB

Benzene

Toluene

0 0 0 0 0 0 0 19 100 (161) 125 14 61 64 123

0 0 0 0 0 0 0 154 100 (257) 119 25 58 50 136

0 0 0 0 0 0 0 263 100 (300) 125 26 61 53 123

0 0 0 0 0 0 0 ND 100 (117) 149 13 64 100 94

Oxygen uptake rates are expressed as percentages of that for catechol taken as protein) are given in parentheses for the relative rates reported as 100%. I The same phenols were tested as those listed in Table 1. c CDHCHD, cis-1,2-Dihydroxycyclohexa-3,5-diene. d ND, Not determined. a

100%.

Absolute activities (as nanomoles

of 02 per minute per milligram of

VOL. 52, 1986

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1379

TABLE 4. Specific catabolic enzyme activities in cell extracts of Alcaligenes sp. strain A175a Assay substrate

Enzyme

cis-1,2-Dihydroxycyclohexa-3,5-diene oxidoreductase 1,2-Pyrocatechase

Enzyme activity for growth substrate: Monochlorobenzene

1,3-DCB

1,4-DCB

Benzene

cis-1,2-Dihydroxycyclohexa-3,5-diene

(584)

(452)

(628)

(703)

Catechol 3-Chlorocatechol 4-Chlorocatechol 3,6-Dichlorocatechol 3-Methylcatechol 3,4-Dimethylcatechol 3,6-Dimethylcatechol

100 (392) 128 29 58 56 0 135

100 (266) 132 29 57 39 0 132

100 (395) 132 29 64 61 0 122

100 (431) 114 15 56 59 0 117

Toluene

NDb 100 (269) 127 20 56 41 0 137

a Enzyme activities are expressed as absolute specific activities in nanomoles per minute per milligram of protein (in parentheses) and as percentages of that for catechol taken as 100%. b ND, Not determined.

growing on 1,4-DCB, suggests the evolution of bacteria with all the degradative genes through the process of natural genetic engineering as proposed by Kellogg et al. (16). In this process, specific genetic information that is present in two or more organisms in a habitat is consolidated in one organism by naturally occurring gene transfer. Although the mechanism of gene transfer has not been studied and consequently cannot be proven, we assume that at least part of the genetic information of the metabolic pathway of 1,4-DCB is located on plasmids, which are subject to interspecies movement (10, 23, 26). This is supported by the observation that cells of Alcaligenes sp. strain A175 lose their ability to mineralize 1,4-DCB after prolonged growth on a medium without 1,4-DCB. Apart from growth on 1,4-DCB, Alcaligenes sp. strain A175 could only use 1,3-DCB, monochlorobenzene, and benzene as sole carbon and energy sources. Its inability to use any of the phenolic compounds, specifically 2,5dichlorophenol, demonstrates the absence of monooxygenases in the metabolic pathway, as was postulated by Ballschmiter and Scholz (2). This is in agreement with the observation of Reineke and Knackmuss (23), who isolated a bacterium, strain WR1306, that utilizes monochlorobenzene

E

~1.0

c

-h\ c

0~~

3,6- dimethylcatechoL

as a sole carbon and energy source, and of de Bont et al. (6), who reported the degradation pathway of 1,3-DCB by an Alcaligenes sp. In addition, the chlorophenols, identified by Ballschmiter and Scholz (2) as intermediates in the degradation of chlorobenzenes, can readily be interpreted as arti-

facts derived from the products of initial dioxygenations of the chlorobenzenes. Because strain A175 showed no activity with 4chlorophenol, it may be concluded that a pathway via an initial dechlorination (halidohydrolysis), as was found by Marks et al. (20) for the degradation of 4-chlorobenzoate by an Arthrobacter species, does not exist. The data from the experiments in which oxygen uptake rates were determined are consistent with a pathway that is mediated by a dioxygenase and an oxidoreductase and that proceeds via 3,6-dichlorocatechol. This is reinforced by the high activity of the enzyme cis-1,2-dihydroxycyclohexa-3,5diene oxidoreductase, found in bacteria which were pregrown on the chlorinated benzenes, and by the detection of 3,6-dichlorocatechol as an intermediate with 1,4-DCB as substrate. A similar pathway has been reported for the degradation of 1,4-DCB in P. putida (Kilbourn and Gibson, Abstr. Annu. Meet. Am. Soc. Microbiol. 1979) and for monochlorobenzene in strain WR1306 (23). Results from oxygen uptake experiments also suggest a high degree of specificity of the initial dioxygenase enzyme. Cells pregrown on 1,4-DCB showed higher uptake rates with 1,2-DCB, 1,3-DCB, and 1,2,4-trichlorobenzene than with 1,4-DCB. No oxygen uptake was observed with chlorinated phenols, benzoate, toluene, xylene, or cresol. The dichlorobenzene dioxygenase enzyme is inducible, as may be concluded from the lack of oxygen uptake by cells pregrown on acetate (data not shown). Strain A175 appar-

2,5 - dimethytmucoruc acid 1

0

30

90 time (min) FIG. 5. Turnover of 3,6-dimethylcatechol and formation of 2,5dimethylmuconic acid by cell extract of Alcaligenes sp. strain A175. Incubation of 1.0 ml of cell extract of strain A175 took place in the presence of 0.3 ml of 40 mM EDTA, 0.3 ml of 33 mM 3,6dimethylcatechol, and 8.4 ml of 40 mM Tris hydrochloride buffer (pH 8.0) at 300C. 60

Cl a

Cl

Cl

cl 0

,

OH 1+OHH OH H

CI

(Cl) COOH COOH

2 OH OH

3

(Cl) d

FIG. 6. Proposed initial steps of 1,4-DCB degradation by Alcaligenes sp. strain A175. (a) 1,4-Dichlorobenzene; (b) 3,6-

dichloro-cis-1,2-dihydroxycyclohexa-3,5-diene; (c) 3,6-dichlorocatechol; (d) (2,5-dichloro)muconic acid; 1, dichlorobenzene dioxygenase; 2, cis-1,2-dihydroxycyclohexa-3,5-diene oxidoreductase; 3, 1,2-pyrocatechase.

1380

APPL. ENVIRON. MICROBIOL.

SCHRAA ET AL.

ently lost this dioxygenase activity during the preparation of the cell extract. Only ortho cleavage of the ring could be detected after the formation of 3,6-dichlorocatechol. The enzyme 1,2pyrocatechase was not very substrate specific. Activities were detected for both methylcatechols and chlorocatechols. It did, however, show a preference for the substituent at the 3-position. No significant activity differences among monochlorobenzene, 1,3-DCB, 1,4-DCB, benzene, and toluene as growth substrates were observed. 1,2-Pyrocatechase catalyzed the transformation of 3,6dimethylcatechol into 2,5-dimethylmuconic acid, while addition of 3,6-dichlorocatechol to cell extracts also resulted in the formation of a muconic acid. The results have been used as a basis for formulating the first three steps in the degradative pathway of 1,4-DCB in Alcaligenes sp. strain A175; these steps are presented in Fig. 6. Incubation of strain A175 with 1,4-DCB, as well as with other aromatic compounds, sometimes resulted in the production in the medium of a red pigment, which turned black over time. This color change preceded an irreversible loss in activity of the cells. As reported by other researchers (3, 9, 11), this color is most probably the result of the accumulation of chlorocatechols which undergo autoxidation and was confirmed in our study by the detection of 3,6-dichlorocatechol in one of the metabolite experiments. During all of our experiments, we were unable to observe a pattern in the occurrence of color formation. Variation of conditions such as concentration of 1,4-DCB, temperature, pH, and oxygen supply did not show a correlation with color formation. One explanation for the additional irreversible inactivity of the bacteria might be the formation of toxic quinone radicals during the polymerization of 3,6-dichlorocatechol. This mechanism as proposed by Bartels et al. (3), in which extradiol cleavage of the catechol results in the formation of lethal acyl halides, is not very likely, however, because of our inability to detect any 2,3-pyrocatechase activity. The inconsistent red pigment formation and catechol accumulation remain puzzling. Research is under way in our laboratory to elucidate the reasons for instability in the metabolism of 1,4-DCB by Alcaligenes sp. strain A175. ACKNOWLEDGMENTS The technical assistance of Wim Roelofsen with the gas chromatography and high-pressure liquid chromatography analyses is gratefully acknowledged. We are grateful to D. A. de Bie, Department of Organic Chemistry, for valuable assistance in the synthesis of intermediates. P.J.C. was funded by the Swiss National Science Foundation (project no. 3.855-0.81). LITERATURE CITED 1. Baker, W., H. F. Bondy, J. Gumb, and D. Miles. 1953. 3:4, 3:5, 3:6-Dimethylcatechol. J. Chem. Soc. 1953:1615-1619. 2. Ballschmiter, K., and C. Scholz. 1980. Mikrobieller Abbau von chlorierten Aromaten. VI. Bildung von Dichlorphenolen und Dichlorbrenzkatechinen aus Dichlorbenzolen in mikromolarer Losung durch Pseudomonas sp. Chemosphere 9:457-467. 3. Bartels, I., H.-J. Knackmuss, and W. Reineke. 1984. Suicide inactivation of catechol 2,3-dioxygenase from Pseudomonas putida mt-2 by 3-halocatechols. Appl. Environ. Microbiol.

47:500-505. 4. Dakin, H. D. 1909. The oxidation of hydroxy derivatives of benzaldehyde, acetophenone and related substances. J. Am. Chem. Soc. 42:477-498. 5. de Bont, J. A. M., A. Scholten, and T. A. Hansen. 1981.

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DNA-DNA hybridization studies of Rhodopseudomonas capsulata, Rhodopseudomonas sphaeroides and Rhodopseudomonas sulfidophila strains. Arch. Microbiol. 128:271-274. de Bont, J. A. M., M. J. A. W. Vorage, S. Hartmans, and W. J. J. van den Tweel. 1986. Microbial degradation of 1,3dichlorobenzene. Appl. Environ. Microbiol. 52:677-680. Dorn, E., and H.-J. Knackmuss. 1978. Chemical structure and biodegradability of halogenated aromatic compounds. Substituent effects on 1,2-dioxygenation of catechol. Biochem. J. 174:85-94. Duff, J. C. 1941. A new general method for the preparation of o-hydroxy-aldehydes from phenols and hexamethylenetetramine. J. Chem. Soc. 1941:547-558. Gibson, D. T., J. R. Koch, C. L. Schuld, and R. E. Kallio. 1968. Oxidative degradation of aromatic hydrocarbons by microorganisms. II. Metabolism of halogenated aromatic hydrocarbons. Biochemistry 7:3795-3802. Grady, C. P. L., Jr. 1985. Biodegradation: its measurement and microbiological basis. Biotechnol. Bioeng. 27:660-674. Haller, H. D., and R. K. Finn. 1979. Biodegradation of 3chlorobenzoate and formation of black color in the presence and absence of benzoate. Eur. J. Appl. Microbiol. Biotechnol. 8:191-205. Hayaishi, O., M. Katagiri, and S. Rothberg. 1957. Studies on oxygenases: pyrocatechases. J. Biol. Chem. 229:905-920. Holding, A. J., and J. M. Shewan. 1974. Genera of uncertain affiliation. Genus Alcaligenes, Castellani and Chalmers 1919, 936, p. 273-275. In R. E. Buchanan and N. E. Gibbons (ed.), Bergey's manual of determinative bacteriology, 8th ed. The Williams & Wilkins Co., Baltimore. Hugh, R., and E. Leifson. 1953. The taxonomic significance of fermentative versus oxidative metabolism of carbohydrates by various gram negative bacteria. J. Bacteriol. 66:24-26. Jenkins, R. O., and H. Dalton. 1985. The use of indole as a spectrophotometric assay substrate for toluene dioxygenase. FEMS Microbiol. Lett. 30:227-231. Kellogg, S. T., D. K. Chatterjee, and A. M. Chakrabarty. 1981. Plasmid-assisted molecular breeding: new technique for enhanced biodegradation of persistent toxic chemicals. Science

214:1133-1135. 17. Kilbane, J. J., D. K. Chatterjee, J. S. Karns, S. T. Kellogg, and A. M. Chakrabarty. 1982. Biodegradation of 2,4,5trichlorophenoxyacetic acid by a pure culture of Pseudomonas cepacia. Appl. Environ. Microbiol. 44:72-78. 18. Kuhn, E. P., P. J. Colberg, J. L. Schnoor, 0. Wanner, A. J. B. Zehnder, and R. P. Schwarzenbach. 1985. Microbial transformations of substituted benzenes during infiltration of river water to

groundwater: laboratory column studies. Environ. Sci. Technol. 19:961-968. 19. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall.

1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. 20. Marks, T. S., A. R. W. Smith, and A. V. Quirk. 1984. Degradation of 4-chlorobenzoic acid by Arthrobacter sp. Appl. Environ. Microbiol. 48:1020-1025. 21. Nozaki, M. 1970. Metapyrocatechase (Pseudomonas). Methods Enzymol. 17A:522-525. 22. Pearson, C. R. 1982. Halogenated aromatics, p. 89-116. In 0. Hutzinger (ed.), The handbook of environmental chemistry, anthropogenic compounds, vol. 3, part B. Springer-Verlag,

New York. 23. Reineke, W., and H.-J. Knackmuss. 1984. Microbial metabolism of haloaromatics: isolation and properties of a chlorobenzenedegrading bacterium. Appl. Environ. Microbiol. 47:395-402. 24. Schraa, G., J. R. van der Meer, A. R. W. van Neerven, P. J. Colberg, and A. J. B. Zehnder. 1985. Microbial transformation of micropollutants in soil systems, p. 315-326. In V. Jensen, A. Kjoller, and L. H. Sorensen (ed.), Microbial communities in soil. Elsevier, London. 25. Schwarzenbach, R. P., W. Giger, E. Hoehn, and J. K. Schneider. 1983. Behavior of organic compounds during infiltration of river water to groundwater. Field studies. Environ. Sci. Technol. 17:472-479.

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