APPLIED AND ENVIRONMENTAL MICROBIOLOGY, JUIY 1994, p. 2330-2338 Vol. 60, No. 70099-2240/94/$04.00+0Copyright C 1994, American Society for Microbiology

Regulation of Chloro- and Methylphenol Degradation inComamonas testosteroni JH5

JULIANE HOLLENDER, WOLFGANG DOTT, AND JOHANNA HOPP*

Fachgebiet Hygiene, Technische Universitat Berlin, 13353 Berlin, Germany

Received 1 December 1993/Accepted 18 April 1994

Comamonas testosteroni JH5 was isolated from a mixed bacterial culture enriched on different chloro- andmethylphenols. The strain completely mineralized a mixture consisting of 4-chlorophenol (4-CP) and4-methylphenol (4-MP). During degradation of the mixture, 4-hydroxybenzyl alcohol, 4-hydroxybenzaldehyde,4-hydroxybenzoic acid, and 4-chlorocatechol were detected as short-lived intermediates. Mineralization of4-CPand that of 4-MP occurred successively and were accompanied by diauxic growth, whereas 4-CP and2-methylphenol were mineralized simultaneously. It was ascertained that neither a reversible enzymeinhibition nor potential toxic intermediates caused the observed diauxie. Some facts support the hypothesisthat the successive degradation of 4-CP and 4-MP is regulated on the level of transcription. C. testosteroni JH5contained a meta-cleaving enzyme when pregrown on 4-CP and the isomeric monomethylphenols. Inactivationof this enzyme in the presence of 3-chlorocatechol was observed.

Biological treatment of wastewater and contaminated soilsinvolves degradation of substrate mixtures rather than degra-dation of single compounds; however, our present understand-ing of the processes which accompany the mineralization ofcomplex mixtures is limited. Methyl- and haloaromatic com-pounds are known to be incompatible growth substrates (31,33). Methyl-substituted aromatic substrates generally are de-graded via meta ring fission catalyzed by catechol 2,3-dioxyge-nases, whereas ring fission of chloroaromates occurs by ortho-cleaving 1,2-dioxygenases (19). If both cleaving enzymes areinduced and if their substrate specificity allows the attack ofboth compounds in the mixture, substrates might be channeledinto the wrong pathway. An accumulation of dead-end metab-olites like 4-carboxymethyl-methylbut-2-en-1,4-olide (methyl-lactones) or 5-chloro-2-hydroxy-6-oxohexa-2,4-dienoic acid(chloro-hydroxymuconic semialdehydes) can result (20, 30,36). 3-Chlorocatechol may cause a reversible inhibition orsuicide inactivation of the meta-cleaving 2,3-dioxygenase (1,18).

Bacteria with the ability to mineralize chloro- or methylaro-mates via unusual degradative pathways, however, are known.Assimilation of 4-methylcatechol was observed via a modifiedortho pathway with Alcaligenes eutrophus JMP 134 (27). Com-plete mineralization of 5-chlorovanillate via meta cleavage wasobserved with a Pseudomonas species. The acylchloride formedduring meta fission undergoes cyclization, which leads todisplacement of the halogen (17). The mechanism, however, isrestricted to ortho standing halogens.The ability to synthesize an unusual degradative pathway for

one special compound does not imply that incompatible sub-strate mixtures will be degraded. Genetic engineering is oneway to obtain organisms with the ability to degrade mixtures ofchloro- and methylaromates. This was shown by in vitroconstruction of a bifunctional ortho cleavage pathway using apatchwork assembly of pathway segments from A. eutrophusJMP 134 in recipient Pseudomonas strain B 13 (28). Anotherway is enrichment by selection against meta-cleaving strains or

* Corresponding author. Mailing address: Fachgebiet Hygiene,Technische Universitat Berlin, AmrumerstraB3e 32, 13353 Berlin, Ger-many. Phone: 49-30-314-27533. Fax: 49-30-314-27575.

induction of suitable enzymes in a single strain by exposure tomethyl- and chloroaromates (25, 33). A third way is mutagen-esis. Recently described Pseudomonas strain JS150, obtainedby mutagenesis of a 1,4-dichlorobenzene-degrading parentorganism, simultaneously degraded chloro- and methylphenolsin the presence of phenol (12). We describe a bacterial strainwhich successively assimilates a mixture of 4-methylphenol(4-MP) and 4-chlorophenol (4-CP) without a cosubstrate aswell as several other mixtures of monomethyl- and monochlo-rophenols. This study intended to analyze the compatibility of4-CP degradation and 4-MP degradation. Possible causes forthe observed diauxie on this substrate mixture were investi-gated. Further experiments were designed to explain degrada-tion inhibitions found during the growth of strain JH5 oncertain mixtures of monochloro- and monomethylphenols.

MATERIALS AND METHODS

All chemicals were of the highest purity grade available.Origin and isolation of strain JH5. Strain JH5 was isolated

from a chlorophenol- and methylphenol-transforming bacte-rial consortium from activated sludge, which was enriched bygrowth on a mixture of different methyl- and chlorophenols inan airlift reactor. The influent contained the following sub-strates in changing compositions: phenol (1.2 mM), the iso-meric monomethylphenols (1 mM), 2,4-dichlorophenol (0.67mM), 2,3-, 2,5-, 2,6-, and 3,4-dimethylphenol (2,3-, 2,5-, and3,4-DMP; 0.8 mM), and 4-CP (0.78 mM). The elimination ratevaried depending on the substrate composition of the influentand the mean residence time in the reactor. A stable operationof the experimental plant was not possible. The bacterialconsortium which was established in the reactor was subcul-tured in our laboratory. It was then grown in batch culture ona mineral salts medium (see "Medium and culture condi-tions") containing 4-CP (0.75 mM) and 4-MP (1 mM) as solesources of carbon and energy. Strain JH5 was isolated on solidmedia consisting of mineral salts medium, 1.5% agar (Oxoid,London, United Kingdom) and 0.58 mM 4-CP, which wasadded as a sterile solution to the autoclaved media. Coloniesgrowing on this phenol agar could be differentiated onlymorphologically by parallel transfers to tryptone soy agar. Forstorage, strain JH5 was resuspended in mineral salts medium

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containing 4-CP and 4-MP. Transfers into fresh medium werecarried out at weekly intervals.Medium and culture conditions. The applied mineral salts

medium contained (per liter of deionized water) 2 g ofNa2HPO4* 2H20, 1 g of KH2PO4, 0.62 g of (NH4)2SO4, and0.06 g of K2SO4. After adjusting the pH to 7.2 to 7.4, thesolution was autoclaved and the addition of several sterilestock solutions followed (per liter of medium): 0.5 ml ofMgSO4- 7H20 (240 g/liter), 0.5 ml of CaCl2 - 2H20 (50 g/li-ter), 1 ml of trace element solution (26), 1.5 ml of 1 MNaHCO3, and 2 ml of vitamin solution (9). Batch culturestudies (using inocula of 2.5, 5, and 10%) were performed witha culture of strain JH5 pregrown on 4-CP (1 mM) to the lateexponential phase. The assays were carried out in 300-mlscrew-cap Erlenmeyer flasks which were filled with 100 to 150ml of medium. Incubation was done in the dark at 25°C on arotary shaker. Samples were taken from duplicate flasks atspecific intervals. Degradation assays with cell suspensions ofstrain JH5 also employed cultures pregrown on 4-CP. Cellswere centrifuged, washed with 50 mM K2HPO4-KH2PO4buffer (pH 7.5), and resuspended in the same buffer.

Characterization and identification of strain JH5. Afterstudying the cell morphology with a microscope (Leitz, Wet-zlar, Germany), Gram staining (modified Hucker method),and oxidase testing (Bactident 13000; E. Merck AG, Darm-stadt, Germany), the isolate JH5 was characterized pheno-typically with 87 physiological tests (16). The resulting testprofiles were compared with a data base by using the methodof numerical identification (16). Cellular fatty acids wereextracted, transmethylated, and analyzed by gas-liquid chro-matography as reported elsewhere (15). Ubiquinones wereisolated from acetone extracts of dry bacterial cells by reverse-phase thin-layer chromatography, slightly modified as describedby Collins (5). They were determined by UV absorption (254nm) and identified by standard ubiquinone extracts fromComamonas testosteroni ATCC 11996T (Q-8).

Tests on substrate specificity. The substrate specificity ofstrain JH5 was tested by incubating the pure culture of JH5(5% inoculum) with several aromatic compounds as the solesources of carbon and energy. The compounds were suppliedin concentrations of 0.25 mM. 2,3-Dichlorophenol, 2,4-dichlo-rophenol, and 2,4,5-trichlorophenol were tested additionally inconcentrations of 0.2, 0.1, and 0.05 mM. Controls containedthe test compound in combination with the degradable mixtureof 4-MP and 4-CP. This setup allowed measurement of co-metabolic degradation as well as evaluation of toxic effects.Sterile controls permitted measurement of abiotic substrateloss. After 14 days of incubation, test assays were stopped andthe optical density (OD), substrate loss, and chloride releasewere determined.

Degradation of monomethyl and monochlorophenols inmixture. Substrate mixtures consisting of one monochlorophe-nol (0.75 mM) and one monomethylphenol (0.75 mM) wereassembled. This resulted in nine different combinations thatwere tested. All monochlorophenols and monomethylphenolswere tested as single compounds (0.75 mM), and a mixturecontaining all of them (0.25 mM per compound) was assem-bled and served as the growth substrate. Mixtures and singlecompounds were supplied as the sole sources of carbon andenergy. They were added as sterile stock solutions to theinoculated (2.5%) mineral salts medium. After 14 days ofincubation, tests were analyzed for substrate degradation,metabolite formation, and chloride release.

Analytical measurements. Cell growth was monitored bymeasuring the OD at 580 nm (OD580) in a spectrophotometer(Spectronic 501; Milton Roy Company, Unterfoehring, Ger-

many). Chloride release was measured at 23°C by potentio-metric detection with an ion-selective Ag-AgCl electrode (11).Substrates and metabolites were analyzed directly in theculture supernatant by high-performance liquid chromatogra-phy (HPLC)-UV diode-array detection. Identification wasdone by comparing the UV spectra and retention times withthose of reference substances. Analysis involved the use of anHP 1090 diode-array detector (Hewlett-Packard Corp., BadHomburg, Germany). A reversed-phase C8 column was used(particle size, 5,um; 5 by 150 mm; Merck) with methanol and0.005 M KH2PO4-H3P04 buffer (pH 2.5; 50:50) as the mobilephase at a flow rate of 1 ml/min. Methyllactones were analyzedby using methanol and the buffer mentioned above mixed atthe ratio of 25:75. Substrates and metabolites were quantifiedby calibration with standards.

Isolation, purification, and identification of metabolites.Metabolites which could not be identified by HPLC-UVdiode-array detection had to be isolated from the culturemedium. The culture medium was centrifuged, and the super-natant was extracted three times with equal volumes of ethylacetate. Combined extracts were dried over MgSO4 and evap-orated to dryness. The metabolites were purified by columnchromatography using silica gel conditioned with hexane.Elution was done with hexane-ethyl acetate (50:50). Gaschromatography-mass spectrometry (GC-MS) spectra fromthe dried eluate were recorded on a GC-MS Varian MAT 44Ssystem with a Cpsil-5 column (Chrompack, Bad Homburg,Germany). The mass spectra were obtained at 70 eV. Nuclearmagnetic resonance (NMR) spectra were recorded on aBruker AM 400 instrument by using dimethyl sulfoxide-d6 asthe solvent and tetramethylsilane as the internal standard.Infrared spectra were determined with a Nicolet Magna IRspectrometer 750. A pellet containing the dispersed sample inexcess KBr was used to measure the spectra.Measurement of oxygen uptake and enzyme assays. Cells of

strain JH5 grown on the appropriate substrates to the lateexponential phase were centrifuged, washed with K2HPO4-KH2PO4 buffer (100 mM; pH 7.5), and resuspended in thesame buffer. By dilution with 3 ml of a 50 mM K2HPO4-KH2PO4 buffer (pH 7.5), the OD580 of the cell suspension wasadjusted to a value of about 1. For protein determination bythe method of Bradford (3), a 1:1 dilution of a cell suspensionwith 0.15 M NaOH was treated with heat for 5 min at 95°C.The oxygen uptake rates were measured at 25°C polarograph-ically with an oxygen electrode (Yellow Springs InstrumentCo., Yellow Springs, Ohio). Cell suspensions were saturatedwith air. After 4 min of constant endogenous oxygen uptake,the reaction was started by injecting the assay substrate to afinal concentration of 0.1 mM. Oxygen uptake was monitoredfor 4 min. The obtained rates were corrected for endogenousconsumption. Activities were expressed as nanomoles of 02uptake per minute per milligram of protein. This rate repre-sents the total activity of methylhydroxylase, phenolhydroxy-lases, catechol dioxygenases, and ring fission enzymes.4-MP methylhydroxylase (EC 1.17.99.1) and catechol 1,2-

dioxygenase (C12 0; EC 1.13.11.1) activities were determinedspectrophotometrically by the methods of Rudolphi et al. (29)and Nakazawa and Nakazawa (22), respectively. Simulta-neously present catechol 2,3-dioxygenase, which interfereswith the determination of catechol 1,2-dioxygenase activity,was destroyed by incubating the crude extract for 5 min withH202 (0.01%). Catechol 2,3-dioxygenase (C23 0; EC1.13.11.2) activity was measured as described by Nozaki (23).The conversion of methylcatechols to muconic acid semialde-hydes was determined by using the spectral data given by Baylyet al. (2). The extinction coefficient for the meta cleavage

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FIG. 1. Successive degradation of 4-CP and 4-MP by strain JH5 pregrown on 4-CP to the late exponential phase (inoculum, 10%). Symbols:*, 4-MP; A, 4-CP; l, Cl-; x, cell density. (A) 4-CP supplied as a single compound; (B, C, and D) 4-CP supplied in mixture with 0.5 (B), 1.0 (C),and 1.5 (D) mM 4-MP.

product of 4-chlorocatechol was 33,000 liters mol-1 cm-' at375 nm. Protocatechuate 2,3-dioxygenase and protocatechuate3,4-dioxygenase (P340; EC 1.13.11.3) activities were deter-mined as described by Crawford (7) and Stanier and Ingraham(32), respectively. Protocatechuate 4,5-dioxygenase (P450; EC1.13.1.8) activity was measured at 410 nm by the method ofOno et al. (24), using simply a 50 mM Tris-acetate buffer (pH9) and omitting an addition of ethanol in the assays. Thereaction mixtures for the activity measurements contained thetest compounds in concentrations of 0.2 mM each and 0.004 to0.4 mg of protein. Crude extracts were prepared by treatingcell suspensions with ultrasonic disintegration (0.5 min, fourtimes, 4°C). The cell debris was removed by centrifugation at65,000 x g (40 min, 4°C), and the resulting supernatant wasstored on ice until use. Protein was determined by the methodof Bradford (3).

Inactivation experiments. Cells grown on 2-MP or 3-MPwere harvested during the late exponential growth phase. Theywere centrifuged, washed, and resuspended in 50 mM Tris-hydrochloride buffer (pH 6.8) containing 1 mM dithiothreitoland 1 mM FeCl2. Cell extracts were prepared as describedabove. Catechol 2,3-dioxygenase inactivation experiments wereperformed by adding 3-chlorocatechol to a final concentrationof 1 mM to the crude cell extract (2.5 mg/ml of protein). Thisreaction mixture was incubated for 30 min at 4°C. Measure-ment of the 2,3-dioxygenase activity then was done. Thereversibility of inactivation was examined by dialyzing thesamples for 20 h twice against 1 liter of the Tris-hydrochloridebuffer (pH 6.8) containing 1 mM dithiothreitol and 1 mM

FeCl2 at 4°C. Control assays were conducted with dialyzed andundialyzed cell extracts.

RESULTS

Characterization and identification of strain JH5. StrainJH5 was found to be gram negative, oxidase positive, andnonfermentative. From dry bacterial cells of strain JH5,ubiquinones with 8 isoprene units in the side chain wereisolated predominantly. Extracted cellular fatty acids werespecified by the number of carbon atoms, double bonds,cyclopropane fatty acids indicated by "c," and, if present, theposition of a hydroxylation. Strain JH5 showed the followingfatty acid profile (area percentages shown in parentheses): for3-OH, 10:0 (4.5), 12:0 (3.0), 15:0 (0.7), 16:1 (24.9), 16:0 (35.1),17:1 (1.1), 17:Oc (10.8), and 17:0 (1.3); for 2-OH, 16:0 (1.4),18:2 (0.6), and 18:1 (16.1). Comparison of 87 physiologicalproperties of JH5 with the data base (14) allowed its identifi-cation as C. testosteroni (data not shown). Results showed ahigh Willcox probability (P > 0.99), low taxonomic distances (d< 0.25), and a difference in no more than 4 characters of 87 incomparison with the data base. The predominant ubiquinoneand fatty acid profiles mentioned were in line with literaturedata (6, 34, 35).4-CP degradation in combination with 4-MP, 4-hydroxyben-

zoic acid (4-HBA), and 2-MP, respectively. All of the subse-quently described experiments were conducted with strain JH5pregrown on 4-CP (1 mM).

Degradation of 4-CP was tested in combination with 4-MP

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0(-)

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FIG. 2. Simultaneous degradation of 4-CP and 2-MP by strain JH5pregrown on 4-CP to the late exponential phase (inoculum, 10%).Symbols: X, 2-MP; A, 4-CP; |, Cl-; X, cell density.

in various concentrations (0.5, 1.0, and 1.5 mM). The assayswere started at a low OD. For comparison, degradation of4-CP was tested in the absence of 4-MP (Fig. 1A). Mineral-ization of the compound took about 24 h. Turnover of 4-CPwas very slow within the first 16 h of incubation. After this lag,the main amount of 4-CP was degraded within 8 h, andstoichiometric amounts of chloride were released. Degrada-tion of 4-CP also took 24 h when supplied in mixture with 0.5mM 4-MP, but in the first hours of incubation, 4-MP wasmineralized exclusively, and then the breakdown of 4-CPfollowed (Fig. 1B). If 4-MP was supplied in higher concentra-tions (1 or 1.5 mM), degradation of 4-CP started with a delayof 12 to 14 h after 4-MP was mineralized (Fig. 1C and D) anda diauxic growth could be observed. In some cases duringsuccessive degradation of 4-MP and 4-CP, formation of tran-sient metabolites was observed. 4-Hydroxybenzyl alcohol,4-HBA, and 4-chlorocatechol were detected as transient inter-mediates in different concentrations (0.2 to 0.02 mM). Inde-pendent of the presence or absence of metabolites, the diauxicgrowth pattern was always observed and the breakdown of4-MP and that of 4-CP always occurred successively. Thediauxie also could be confirmed on a mixture consisting ofacetate and 4-CP, with acetate degradation occurring first(data not shown). In contrast, 4-MP-pregrown strain JH5degraded 4-MP first and then acetate (data not shown).

Mixtures consisting of 4-CP and 2-MP (Fig. 2) or 4-CP and3-MP (data not shown) were mineralized simultaneously. Sinceno UV-detectable metabolites accumulated and chloride wasreleased in stoichiometric amounts, a complete mineralizationof these mixtures can be assumed.

Additional experiments with the substrate mixture of 4-CPand 4-MP were conducted with strain JH5 either in theexponential growth phase or under stationary growth condi-tions. Cells of JH5 in the logarithmic phase of growth on 4-CPwere provided with different additions. Figure 3A shows thecourse of 4-CP degradation during logarithmic growth. 4-CPwas mineralized with a steady turnover rate. An additionalsupplement of 4-MP (Fig. 3B) or 4-HBA (Fig. 3C) in aconcentration of about 1 mM caused a significant delay of the4-CP mineralization. Both assays initially showed a phase ofsimultaneous degradation of 4-CP and 4-HBA (Fig. 3C) or4-CP and 4-MP (Fig. 3B). Degradation of the chlorophenolthen stopped until 4-HBA or 4-MP was completely mineral-

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Time [h]FIG. 3. Influence of 4-MP and 4-HBA on 4-CP degradation during

exponential growth of strain JH5. (A) Course of 4-CP degradationduring exponential growth of JH5; (B and C) course of 4-CP degra-dation when 4-MP and 4-HBA were supplied in addition. Symbols: *,4-MP; A, 4-CP; X, 4-HBA; |, Cl-; X, cell density.

ized. After 2 h of incubation in the assay corresponding to Fig.3B, a methylhydroxylase was induced as measured by enzymeassays.

In contrast to this, assays conducted with concentrated cellsuspensions of strain JH5 (OD580, 3.14) showed simultaneousmineralization of 4-CP and 4-MP (Fig. 4). Growth was notobserved. Despite the fact that a methylhydroxylase activitycould not be determined, 4-hydroxybenzaldehyde and 4-HBAwere sometimes detected as transient metabolites in concen-trations of 0.01 to 0.04 mM.Enzyme activities and oxygen uptake rates of whole cells of

JH5. Cells grown on 4-MP oxidized this carbon source as wellas 4-HBA (Table 1). These cells, however, were not induced tooxidize 4-CP nor any of the other isomeric monochloro- ormonomethylphenols. In addition, cells grown on the mixture of4-MP and 4-CP and harvested during 4-MP breakdown pro-duced high uptake rates with 4-MP, whereas no activity wasfound with 4-CP. Proposed intermediates of 4-MP breakdownlike 4-hydroxybenzyl alcohol, 4-hydroxybenzaldehyde, 4-HBA,and protocatechuate also were oxidized.

In contrast, cells grown on 4-CP were highly induced tooxidize this carbon source as well as 4-MP. In addition, theother isomeric monomethylphenols were oxidized. To a lesser

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00 20 40 60 80 100o 1,20

Time [min]

FIG. 4. Degradation of 4-CP and 4-MP by concentrated cell sus-pension of JH5 (OD580, 3.14) pregrown on 4-CP. Symbols: *, 4-MP;A, 4-CP. Growth was not observed.

extent, there also was a potential to oxidize the isomericmonochlorophenols and some intermediates of 4-MP break-down.

Cells grown on the mixture of 4-CP and 4-MP and harvestedduring 4-CP breakdown produced higher uptake rates on4-MP than on 4-CP. In addition, these cells were induced tooxidize the intermediates of 4-MP breakdown.

Parallel experiments with cell extracts of strain JH5 wereconducted to evaluate the presence of certain enzymes in-volved in degradation of monochloro- and monomethylphe-nols. A methylhydroxylase was induced in cell extracts ofcultures exposed to 4-MP but not in those exposed to 4-CP.Independent of the growth substrate (4-CP, 4-MP, 3-MP, and2-MP) and independent of the growth phases from which cellswere harvested, a catechol 1,2-dioxygenase activity was notdetected. Cultures grown in 4-CP and 4-MP contained acatechol 2,3-dioxygenase (Table 2), which was able to turn over4-methylcatechol and 4-chlorocatechol but not 3-chlorocat-echol. A protocatechuate 2,3-dioxygenase or 3,4-dioxygenaseactivity was not detected. In cell extracts from 4-MP-pregrowncultures, a protocatechuate 4,5-dioxygenase was induced. The

TABLE 1. Oxygen consumption by whole cells of strain JH5

Oxygen consumed (nmol/min/mg of protein)after growth ona:

Test substrate(0.1 mM/substrate) 4-CP + 4-MP

4-CP 4-MP Growth Growthphase 1 phase 2

2-MP 332 0 NDb ND3-MP 306 0 ND ND4-MP 547 1,045 658 5422-CP 204 1 ND ND3-CP 135 0 ND ND4-CP 567 0 1 1974-Hydroxybenzyl alcohol 158 ND 520 4384-Hydroxybenzaldehyde 126 ND 374 3014-HBA 4 528 209 181Protocatechuate 0 ND 114 83

a Oxygen consumption and protein concentrations were determined as de-scribed in the text.

b ND, not determined.

TABLE 2. Specific activities of 2,3-dioxygenases in cell extract ofstrain JH5

Sp act (U/mg of protein)Enzyme assayed and assay substrate after growth on:

4-MP 4-CP

Catechol 2,3-dioxygenase4-Methylcatechol 0.494 0.4194-Chlorocatechol 0.135 0.1733-Chlorocatechol a

Protocatechuate 2,3-dioxygenaseProtocatechuate

, no activity was measured.

enzyme exhibited a specific activity of 0.422 U/mg of proteinwith protocatechuate as the assay substrate.

Degradation of monomethyl- and monochlorophenols inmixtures. The biodegradability of 10 different mixtures ofmonomethyl- and monochlorophenols as well as the biode-gradability of the single compounds was studied (Fig. 5).Complete breakdown was recorded when about 90% of theexpected amount of chloride was released and no HPLC-UV-detectable metabolites were measured. C. testosteroni JH5 wasable to mineralize 4-CP and the three isomeric monomethyl-phenols, whereas 2-CP and 3-CP persisted (Fig. 5). Mixturesconsisting of 4-CP and either of the three isomeric mono-methylphenols were completely degraded. In addition, 4-MPwas mineralized in combination with 2-CP or 3-CP. In theseassays, 3-CP was left nondegradable (turnover, <6%), whereas2-CP was degraded partially (turnover, 30%), obviously bycometabolism. 2-MP and 3-MP persisted in the presence of3-CP and were degraded only to a small extent (2-MP, 37%;3-MP, 10%) in combination with 2-CP. None of the com-pounds was degraded in a mixture consisting of the isomericmonochloro- and monomethylphenols.

Inactivation of catechol 2,3-dioxygenase. Cells of strain JH5

3-MP4-MP2-CP3-CP4-CP

2-P + 2-MP2-OP + 3-MP2-OP + 4-MP3-OP + 2-MP3-P + 3-MP3-OP + 4-MP4-OP + 2-MP4-OP + 3-MP4-CP+ 4-MP

2-/3-/4-CP + 2-13-/4-MPO 20 40 60 80 100

degradafion, release of chloride [%]FIG. 5. Extent of 2-CP, 3-CP, 4-CP, 2-MP, 3-MP, and 4-MP (0.75

mM) degradation by strain JH5 detected after 14 days of incubation.Substrates were supplied as single compounds and as mixtures asshown in the graphic. Symbols: _, extent of Cl- release; El1,degradation (percent) of monomethylphenol; lM, degradation ofmonochlorophenol.

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TABLE 3. Reversibility of inactivation of catechol 2,3-dioxygenase after treatment with 3-chlorocatechol

Activity with the test substrate:Cell extract of

strain JH5 Treatment" Catechol 4-Methylcatecholgrown on': Without After Without After

dialysis dialysisd dialysis dialysise

2-MP None (control) 100 (2.16)b 97 100 (0.19) 1003-Chlorocatechol (1 mM) <1 11 <1 15

3-MP None (control) 100 (1.81) 103 100 (0.16) 1123-Chlorocatechol (1 mM) <1 10 <1 15

aCell extracts were stored on ice for 20 h.b Treatment with 3-chlorocatechol was for 30 min.' Control values without dialysis were taken as 100% (specific activities in units per milligram of protein are in parentheses). Other values are percentages of control

values.dDialysis was against 50 mM Tris-HCI buffer (pH 6.8) supplemented with 1 mM dithiothreitol and 1 mM FeCI2.

pregrown in 2-MP and 3-MP exhibited the activity of acatechol 2,3-dioxygenase, but the presence of a catechol 1,2-dioxygenase could not be determined. Catechol 2,3-dioxygen-ase in extracts of cells pregrown in 2-MP and 3-MP wascompletely inactivated by 3-chlorocatechol at a concentrationof 1 mM (Table 3). After dialysis, the inactivated cell extractsexhibited 2,3-dioxygenase activities of 10 and 15% on catecholand 4-methylcatechol, respectively.

Substrate specificity of strain JH5 and characterization ofmetabolites. Strain JH5 transformed a variety of organiccompounds (Table 4). Benzoate and some hydroxybenzoateswere completely degraded. Neither the supplied methyllac-tones nor the tested dichloro- and trichlorophenols wereattacked by strain JH5. The latter compounds proved to havea toxic effect when supplied in concentrations higher thanthose shown in Table 4. Complete mineralization was detected

with 2,3-DMP and 4-chloro-3-methylphenol. 2,5-DMP and3,5-DMP were degraded partially in cometabolism with 4-CPand 4-MP only. The other tested methylphenols were trans-formed to UV-detectable dead-end metabolites (Fig. 6). Theproducts were extracted from the culture fluid and purified bycolumn chromatography as described in Materials and Meth-ods. 2,4-DMP and 3,4-DMP were transformed completely toUV-detectable dead-end metabolites. The two metaboliteswere identified as 2-methyl-4-HBA (Fig. 7A) and 3-methyl-4-HBA (Fig. 7B), respectively, by structure elucidation. Theirmass spectra have similarities as shown in Fig. 7A and B.Significant fragments were assigned as shown in Table 5. Dataobtained from the 1H-NMR spectra (Table 6) confirm thesuggested structures of both compounds. Although the OHand COOH groups were not apparent in the 1H-NMR data,their presence was ascertained by infrared spectroscopy reveal-

TABLE 4. Spectrum of substrate utilization tested for strain JH5

Substrate (0.25 mM) Degradation'

Benzoic acid.............................................................................2-HBA ......................................................................................3-HBA ......................................................................................4-HBA ....................................................................................2,4-Dihydroxybenzoic acid.....................................................Protocatechuate .......................................................................Phenol.......................................................................................2,3-DMP ...................................................................................2,4-DMP ...................................................................................2,5-DMP ...................................................................................2,6-DMP ...................................................................................3,4-DMP ...................................................................................3,5-DMP...................................................................................2,3,5-Trimethylphenol .............................................................2,4,6-Trimethylphenol .............................................................2,3-Dichlorophenol (0.1 mM)................................................2,4-Dichlorophenol (0.2 mM)................................................2,5-Dichlorophenol .................................................................2,6-Dichlorophenol .................................................................3,4-Dichlorophenol (0.1 mM)................................................2,4,5-Trichlorophenol (0.05 mM)..........................................2,4,6-Trichlorophenol .............................................................4-Chloro-3-methylphenol .......................................................2-Chloro-4-methylphenol .......................................................4-Bromophenol........................................................................2-Methyllactone .......................................................................3-Methyllactone .......................................................................4-Methyllactone .......................................................................

+ Met B

+ Met A

± Met D

+ MetC

OH OH .1

CH3 COOH

OHCH3

OHCH3

CH3

OH

CH3CH3

OHCI

CH3

OHCH3 CH3

CH3

OH

,CH3

OH

CH3

CH3

OH

CH3a

+, complete degradation; -, no degradation; +, partial degradation; Met A,B, C, and D, conversion to dead-end metabolites (mass spectra shown in Fig. 7).

FIG. 6. Degradation of methylphenols by strain JH5. Symbols: *1,identified intermediate; *2, identified dead-end metabolite.

patway ? Co2

1- C02 . cr

OH *2CH3

COOH

OH *2

-2CH3

COON

OH *2a

COOH

OH *2CH3 CH3

0O

COON

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2336 HOLLENDER ET AL.

OH d

eH Hc

0~~~~f H CHb

COOH a

77 107106

53 1 8 105-63 76 -8 2

L1 l. -.1 .117 121.

1 00

OH d

eH)A CH,C

f H <-Hb

COOH a

77

51 79

Ii, L6.2.L7. jI 9I0

107

105 108_1 I

134k35 152_

151

136

1 .1

C100-

80-60-

48-

28-153

*I-1-150

152

I134 6

60 80 100 120 140

53

D

88-

68-40-

20-

160

m/z

OH

dH0ci

cH HbCOOH a

II

155

63

62 127 54

- 4 91I 1 96065 i98I110

0 ,0 2,

80 100 120 140

OH d

cH3C CH3 c

HNbCOOH 0

77 91

p3 65 107

-63 78 93

L II.,6,i1.i..;7.. .....1.3.L,

121

1 680 1 0 1 20

149

122

1. 1101

172

157

173

160 180

1 66

1671 65

FIG. 7. Mass spectra of 2-methyl-4-HBA (A), 3-methyl-4-HBA (B), 3-chloro-4-HBA (C), and 3,5-dimethyl-4-HBA (D).

ing characteristic band maxima at 3,100 to 3,600 cm-' and1,689 cm-', the valence vibrations of 0-H and aryl-C)0,respectively.

2-Chloro-4-MP was transformed stoichiometrically to a UV-detectable dead-end metabolite, which was identified as3-chloro-4-HBA by GC-MS and 'H-NMR measurements. Fig-ure 7C shows the mass spectrum of the compound. The ionsignals are accompanied by smaller n + 2 peaks in ratiosconsistent with the presence of a single chlorine atom. Thesignificant fragments formed were assigned as shown in Table5. Data obtained from the 1H-NMR measurement confirm theproposed structure. The resonance lines and their assignmentsare given in Table 6.

2,4,6-Trimethylphenol was degraded only partially to aUV-detectable dead-end metabolite, which was identified as3,5-dimethyl-4-HBA by 'H-NMR and GC-MS measurements.Characteristic ion signals occurring in the spectrum (Fig. 7D)

TABLE 5. Mass spectral data of metabolites A, B, C, and D

Compound m/z Assignment

2-Methyl-4-HBA (metabolite A) 152 M+134 M+-H20107 M+-COOH

3-Methyl-4-HBA (metabolite B) 152 M+135 M+-OH107 M+-COOH77 [C6H5+J

3-Chloro-4-HBA (metabolite C) 172 M+155 M+-OH127 M+-COOH99 M+-COOH-CO

3,5-Dimethyl-4-HBA (metabolite D) 166 M+149 M+-OH121 M+-COOH91 M+-COOH-CH3-CH377 [C6H5+1

are interpreted as shown in Table 5. The integral of theresonance lines of the 'H-NMR spectrum (Table 6) corre-sponds to the expected 10 protons. Their chemical shifts,coupling patterns, and assignments are given in Table 5.Comparable 'H-NMR and mass spectral data for the isolatedmetabolites were not available from the literature.

DISCUSSION

C. testosteroni JH5, described in this article, completelymineralized a mixture consisting of 4-CP and 4-MP, although

TABLE 6. 1H-NMR spectral data of metabolites A, B, C, and D

Compound' Proton Signal Description'(ppM)b Dsrpin

2-Methyl-4-HBA a 10.03 1H, s(metabolite A)

b 2.45 3H, sc 6.63 1H, se 6.62 1H,d, Jef = 9.0f 7.74 1H, d, Jfe = 9.5

3-Methyl-4-HBA b 7.67 1H, d, Jbf = 2.0(metabolite B)

c 2.13 3H, se 6.82 1H, d, Jef = 8.0f 7.61 1H, q, Jfe = 8.0, Jfb = 2.0

3-Chloro-4-HBA b 7.83 1H, d, Jbe = 2.0(metabolite C)

d 7.02 1H, d, Jde = 8.5e 7.73 1H, q, Jed = 8.5, Jeb = 2.0

3,5-Dimethyl-4-HBA a 12.30 1H, s(metabolite D)

b 7.52 2H, sc 2.17 6H, sd 9.00 1H, s

For diagrams of the compounds, refer to Fig. 7.b Chemical shifts are giveni as parts per million downfield from tetramethylsi-

lane in dimethyl sulfoxide-d6.' Singlet, doublet, and quartet are abbreviated s, d, and q, respectively;

coupling constants (J) are given in hertz.

A-100

810680

jgr 20

C a

. B_ e

a) IB-!

60-

40-

20-

140 160 180

4

i

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COOXIDATIVE DEGRADATION OF SUBSTITUTED PHENOLS 2337

the compounds are known to be incompatible growth sub-strates. The literature indicates that mineralization of incom-patible substrates is achieved by induction of a single type ofaromatic ring cleavage (28, 30). It was shown, however, that thecoexistence of meta and ortho pathways is possible (33, 25).Unproductive pathways are prevented either by a controlledinduction mechanism or the absence of an active catechol2,3-dioxygenase under certain conditions (25). C. testosteroniJH5 degrades 4-CP and 4-MP successively (Fig. 1 and 2). Thus,the separation of their degradations in time presents a newmechanism to achieve compatibility. Furthermore, results in-dicate that C. testosteroni JH5 employs a novel mechanism in4-CP degradation. 4-CP-pregrown cells contained a 2,3-dioxy-genase which was able to convert 4-chlorocatechol. A 1,2-dioxygenase activity could not be detected in these cells. Thus,the complete degradation of 4-CP via meta cleavage of 4-chlo-rocatechol is postulated for the first time in this article.The possible causes for the observed successive degradation

of 4-CP and 4-MP were investigated. An inhibitory or toxiceffect of the intermediates of 4-MP breakdown causing thediauxie on the substrate mixture can be excluded positively.Strain JH5 in the exponential phase of growth on 4-CP, whichwas supplied with additional 4-MP or 4-HBA, initially de-graded both compounds simultaneously (Fig. 2B and C). Withinduction of the methylhydroxylase and other enzymes of thepreferred degradation pathway of 4-MP, the pattern of sub-strate utilization changed. Degradation of 4-CP stopped until4-MP or 4-HBA was completely degraded. The observedrepression of 4-CP breakdown might be caused by 4-HBA,4-MP, or their catabolites.

In addition, a simultaneous mineralization of 4-CP and4-MP was found in assays conducted with concentrated cellsuspensions of strain JH5 pregrown in 4-CP. In these cellsuspensions, induction of a methylhydroxylase could be ex-cluded by enzyme measurements. Detection of 4-HBA and4-hydroxybenzaldehyde after the start of the experiment justaccounts for the presence of the methylhydroxylase on a verylow level. The experiment indicates that C. testosteroni JH5 isable to mineralize 4-MP via a second pathway. This resultagrees well with oxygen uptake rate measurements. Oxygenuptake rates showed that cells grown on 4-CP oxidized 4-MP aswell as 2-MP and 3-MP, whereas 4-MP-grown cells exhibitedno activity on 4-CP or on the isomeric methylphenols (Table1). The presence of a methylhydroxylase in 4-MP-pregrowncells of JH5 and the lack of this enzyme in 4-CP-grown strainJH5 support this assumption. The pathway induced initially by4-MP methylhydroxylase probably is preferred, and inductionof this enzyme occurs whenever 4-MP is present in mixtures.Degradation of 4-MP then proceeds via 4-HBA and protocat-echuate. The latter compound was meta cleaved by a 4,5-dioxygenase as described for Pseudomonas testosteroni (8). Theresults obtained suggest that the active methylhydroxylaseexclusively oxidizes aromatic methyl substituents in the paraposition (Table 4; Fig. 6). Results show that methyl groups inother positions remained intact, as also was shown for Pseudo-monas putida NCIB 9869 (21). Thus, multiple methylatedphenols with a para-methyl group were transformed to theappropriate 4-HBAs, which persisted and accumulated in theculture liquid. P. putida NCIB 9866, in contrast, provides adifferent enzyme activity. 2,4-DMP mineralization via 4-hy-droxyisophthalate as an intermediate shows that the methylgroups undergo oxidation in succession (4). Since strain JH5completely mineralized 2-MP, 3-MP, and 2,3-DMP, one canassume that the compounds were attacked initially by aphenolhydroxylase. The presence of a 2,3-dioxygenase in2-MP- or 3-MP-pregrown JH5 cells indicates that degradation

occurs by meta cleavage. Other multiply methylated phenolswith methyl groups in ring position 5 or 6 were not or onlypartially degraded. For some of these compounds, completemineralization via ortho ring fission has been described in theliterature (10, 13, 14).

Despite the fact that C. testosteroni JH5 was able to use ameta pathway for 4-CP degradation, incompatibilities on cer-tain substrate mixtures, however, could not be prevented.Degradation of 2-MP and 3-MP was inhibited in the presenceof 2-CP or 3-CP. Since 4-MP proved to be degradable incombination with 2-CP or 3-CP, a toxic effect of the providedchlorophenol concentration was excluded. The inhibition canbe explained by the assumption that the phenol hydroxylaseinduced on 2-MP and 3-MP reacts with 2-CP and 3-CP,forming 3-chlorocatechol, which in turn reacts with the in-duced 2,3-dioxygenase. The latter reaction is irreversible, asshown by our results (Table 3). No such pathway incompati-bilities were found with Pseudomonas strain JS150. The straindegrades mixtures of 2-CP, 3-CP, 2-MP, and 3-MP in thepresence of phenol (12). In contrast to strain JS150, C.testosteroni JH5 contains no ortho-cleaving enzymes, whichmight explain its restricted degradation capacity.

ACKNOWLEDGMENTSThis research was supported by the Deutsche Forschungsgemein-

schaft.We thank K.-H. Engesser for providing 2-, 3-, and 4-methyllactone,

P. Kampfer for identifying pure cultures, and U. Wiesmann forproviding the mixed culture as well as G. Hohne for the GC-MSmeasurements.

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inactivation of catechol 2,3-dioxygenase from Pseudomonas putidamt-2 by 3-halocatechols. Appl. Environ. Microbiol. 47:500-505.

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4. Chapman, P. J. 1972. An outline of reaction sequences used forthe bacterial degradation of phenol compounds, p. 17-55. In P. J.Chapman and S. Dagley (ed.), Degradation of synthetic organicmolecules in the biosphere. National Academy of Sciences, Wash-ington, D.C.

5. Collins, M. D. 1985. Isoprenoid quinone analysis in bacterialclassification and identification, p. 267-287. In M. Goodfellow andD. E. Minnikin (ed.), Chemical methods in bacterial systematics.Academic Press Ltd., London.

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