APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1990, p. 3842-3850 Vol. 56, No. 120099-2240/90/123842-09$02.00/0Copyright C) 1990, American Society for Microbiology

Degradation of Mono-, Di-, and Trihalogenated Benzoic Acidsby Pseudomonas aeruginosa JB2

W. J. HICKEYt AND D. D. FOCHT*

Department of Soil and Environmental Science, University of California, Riverside,Riverside, California 92521

Received 4 April 1990/Accepted 28 September 1990

Pseudomonas aeruginosa JB2 was isolated from a polychlorinated biphenyl-contaminated soil by enrichmentculture containing 2-chlorobenzoate as the sole carbon source. Strain JB2 was subsequently found also to growon 3-chlorobenzoate, 2,3- and 2,5-dichlorobenzoates, 2,3,5-trichlorobenzoate, and a wide range of other mono-and dihalogenated benzoic acids. Cometabolism of 2,4-dichlorobenzoate was also observed. Chlorocatecholswere the central intermediates of all chlorobenzoate catabolic pathways. Degradation of 2-chlorobenzoate wasrouted through 3-chlorocatechol, whereas 4-chlorocatechol was identified from the metabolism of both 2,3- and2,5-dichlorobenzoate. The initial attack on chlorobenzoates was oxygen dependent and most likely mediated bydioxygenases. Although plasmids were not detected in strain JB2, spontaneous mutants were detected in 70%of glycerol-grown colonies. The mutants were all of the following phenotype: benzoate+, 3-chlorobenzoate+,2-chlorobenzoate-, 2,3-dichlorobenzoate-, 2,5-dichlorobenzoate-. While chlorocatechols were oxidized by themutants at wild-type levels, oxidation of 2-chloro- and 2,3- and 2,5-dichlorobenzoates was substantiallydiminished. These findings suggested that strain JB2 possessed, in addition to the benzoate dioxygenase, ahalobenzoate dioxygenase that was necessary for the degradation of chlorobenzoates substituted in the orthoposition.

Chlorinated benzoic acids are introduced into the environ-ment chiefly through two sources, namely, herbicides andpolychlorinated biphenyls. The latter chemicals give rise tochlorobenzoates (CBas) by microbial cometabolic decompo-sition through the meta-cleavage pathway (12, 32, 36).However, bacteria that cometabolically attack polychlori-nated biphenyls accumulate CBas because they are unable togrow on them. CBa degraders, therefore, are essential forcomplete aerobic mineralization of polychlorinated biphe-nyls.Many investigators have reported the isolation of 3-CBa-

utilizing (5, 6, 11, 15, 18, 20, 28, 39) and 4-CBa-utilizing (1,22, 23, 27, 30, 37) bacteria. The metabolism of these CBashas been well defined and shown to proceed through 3-chlo-rocatechol and 4-hydroxybenzoate, respectively. The isola-tion of 2-CBa-degrading bacteria has also been described bya number of researchers (2, 8, 9, 17, 33, 35, 40). Growth ofbacteria on dichlorobenzoates (DCBas) is limited to 2,4-DCBa (17, 37, 41), 2,5-DCBa (2), and 3,5-DCBa (18). To thebest of our knowledge, an organism that grows on any otherisomeric DCBa or on a trichlorinated benzoic acid (TCBa)has not been isolated.The degradation of 2-CBa has been reported to proceed by

two routes. The most common intermediate is catechol (8,10, 35, 40), postulated to be formed by the spontaneousremoval of chlorine and CO2 as the result of a 1,2-dioxygen-ase attack (8, 10). However, in one of these cases (35),chloromuconic acid was also identified, which raises thepossibility that 3-chlorocatechol may also be formed by a1,6-dioxygenase attack. 2,3-Dihydroxybenzoate has alsobeen identified as a product of 2-CBa metabolism (9) but isbelieved to be a dead-end metabolite (10).

* Corresponding author.t Present address: Department of Soil Science, University of

Wisconsin, Madison, Madison, WI 53706.

Metabolism of 2,4-DCBa generally involves completechloride removal prior to ring cleavage via a reductivedechlorination to 4-CBa followed by a hydrolytic dechlori-nation to 4-hydroxybenzoate (37, 41). In contrast, 3,5-DCBawas degraded to 3,5-dichlorocatechol, which then under-went ring cleavage to give a dichlorinated muconic acid (17).The genetic basis of CBa degradation has been investi-

gated in several organisms. Plasmid-borne genes have beenidentified for 3-CBa and 3,5-DCBa degradation (3-5, 13, 38)and implicated for 2-CBa degradation (33). The degradationof 4-CBa, on the other hand, has been reported to bedirected by either chromosomal (31) or plasmid-coded (32)genes.

This report describes the isolation and characterization ofPseudomonas aeruginosa JB2, which used five differentCBas as growth substrates, specifically, 2-CBa, 3-CBa,2,3-DCBa, 2,5-DCBa, and 2,3,5-TCBa. Strain JB2 also useda wide range of other mono- and trihalogenated benzoates asgrowth substrates. The novel substrate range of strain JB2therefore offered an opportunity to delineate two previouslyunreported metabolic pathways, namely, 2,3- and 2,5-DCBa,and examine how metabolism of a range of CBas is coordi-nated within a single organism.

MATERIALS AND METHODS

Isolation and taxonomic identification. P. aeruginosa JB2was isolated by standard enrichment culture techniques,using an inoculum of polychlorinated biphenyl-contaminatedsoil obtained from Fontana, Calif. A mineral salts medium(MSM) containing 2-CBa (3.2 mM) as the sole carbon sourcewas used for enrichment and purification of the culture. TheMSM contained K2HPO4 (10 mM), NaH2PO4 (3 mM),(NH4)2SO4 (10 mM), and MgSO4 (1 mM). Trace elementswere added to MSM to give the following final concentra-tions (in milligrams per liter): CaSO4, 2; FeSO4 7H20, 2;MnSO4- H20, 0.2; CuSO4, 0.2; ZnSO4. 7H20, 0.2;

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CoS04 * 7H20, 0.1; NaMoO4 - 2H20, 0.1; H3BO3, 0.05. Theisolate was a gram-negative rod, motile by a single polarflagellum, oxidase positive, catalase negative, arginine dihy-drolase positive, and fluorescent on both King's A andKing's B media, produced nitrite from nitrate, and did notferment glucose. Cofactors were not necessary for growth inMSM. The isolate also grew on tryptic soy medium contain-ing 1,10-phenanthroline (100 ,ug ml-'), a trait established(23) as characteristic of P. aeruginosa. The isolate wassubsequently identified as P. aeruginosa by standard taxo-nomic criteria (34) and designated strain JB2.

Culture conditions. Strain JB2 was routinely grown inMSM supplemented with 3.2 mM either 2- or 3-CBa. Whenstrain JB2 was cultured on 2,3- or 2,5-DCBa (3.2 mM) or2,3,5-TCBa (2.2 mM), the concentrations of K2HPO4 andNaH2PO4 in MSM were increased proportionally to give afinal concentration of 50 mM total phosphate. This increasein buffer capacity was required to compensate for the greaterHCI production.Growth measurements. Growth of strain JB2 cultures was

routinely monitored by measuring A546 with a Uvikon model860 UVIVIS spectrophotometer (Kontron Instruments, Zu-rich, Switzerland). A culture A546 of 0.4 was determined tocorrespond to 100 p,g of cells (dry weight) ml-'. Thisrelationship was used to convert absorbance readings tobiomass.To define the range of substituted benzoates that strain

JB2 used for growth, a 1% inoculum of a 2-CBa-grownculture was added to 100 ml ofMSM containing 500 ,ug of thetarget substrate ml-'. Also tested as growth substrates forstrain JB2 (at 100 ,ug ml-') were phenol; 2-, 3-, and 4-chlo-rophenol; and 2,3-, 2,4-, 2,5-, 2,6-, 3,4-, and 3,5-dichlorophe-nol. The substrate was determined to support growth ofstrain JB2 if, after a 20-day incubation, the culture had ameasurable increase in absorbance.

Chemicals. 4-Chlorocatechol was obtained from HelixBiotech Corp., Richmond, British Columbia, Canada, while3-chlorocatechol was supplied courtesy of F. Higson, Uni-versity of California, Riverside. 2,3,5-TCBa was obtainedfrom Trans World Chemicals Inc., Chevy Chase, Md. Bo-vine serum albumin, NADH, and flavin adenine dinucleotidewere purchased from Sigma Chemical Co., St. Louis, Mo.All other chemicals were obtained from Aldrich ChemicalCo., Inc., Milwaukee, Wis.GC. Routine metabolite analysis was performed on a

Hewlett-Packard (Palo Alto, Calif.) model 5890 gas chro-matograph (GC) fitted with a DB-5 megabore column (J + WScientific, Folsom, Calif.) and flame ionization detector.Helium was used as the carrier gas at a flow rate of 17 mlmin-'. The temperature program increased from 100 to220°C at a rate of 10°C min-1, while the injector and detectortemperatures were maintained at 240 and 300°C.GC-mass spectroscopy. A Finnigan (San Jose, Calif.)

model 4000 high-resolution mass spectrometer operated atan electron energy of 70 eV was interfaced with a Hewlett-Packard model 5790 GC fitted with a DB-5 capillary column.The GC was operated by using a temperature program risingfrom an initial temperature of 50 to 250°C at 10°C min-' at aflow rate of 1 ml min-1. The injector was maintained at2400C.

Chloride determination. Inorganic chloride was deter-mined turbidimetrically by measuring AgCl precipitation.Samples (1 ml) were acidified with 10 ,ll of 10 N H2SO4 andcentrifuged (5 min at 1,500 x g) to remove material thatprecipitated due to acidification alone. Each sample andstandard was zeroed against itself at 525 nm on a Uvikon

model 860 UVNVIS spectrophotometer (Kontron Instru-ments) to minimize background variation. Precipitation ofAgCl was then measured by adding 10 ,ul of 0.1 M AgNO3 (in5 M H3PO4) and immediately reading the sample A525.Chloride was quantified by reference to a standard curve thatwas linear from 0.1 to 3.2 mM. Samples containing >3 mMchloride (e.g., those from incubations with DCBas orTCBas) were diluted 1:2 in MSM and then analyzed asdescribed above. Blanks, consisting of MSM alone, werefree of interferences due to precipitation of medium compo-nents.

Protein determination. Protein content of resting cell sus-pensions was determined by using the biuret method (25) andquantified by reference to a calibration curve constructed byusing bovine serum albumin as a standard.

Aerobic resting cell incubations. One liter of cells, grown tolate log phase (A546 of ca. 0.3) in 2.5-liter Fernbach flasks,was harvested by 20-min centrifugation at 12,000 x g, usinga Beckman JA-10 rotor and Beckman model J2-21 centrifuge(Beckman Instruments Inc., Fullerton, Calif.). After twowashes with 50 mM potassium phosphate buffer (pH 7.5),cells were resuspended in phosphate buffer to a final densityof 200 mg (wet weight) of cells ml-'. Incubations werecarried out in 5 ml of 50 mM phosphate buffer (pH 7.5), towhich substrates were added to give a final concentration of500 jig ml-'. Metabolism was terminated at specific intervalsby adding 100 ,l of 10 N H2SO4.

Anaerobic resting cell incubations. Cells were prepared foranaerobic incubations by the same protocol outlined abovefor aerobic incubations. Phosphate buffer (50 mM; pH 7.5)was boiled for 20 min and then allowed to cool while astream of N2 was sparged into the liquid. Flasks (50 ml) wereflushed with N2 prior to the addition of 2 ml of buffer.Teflon-coated seals were then affixed to the flasks by usingcrimp caps (Wheaton Scientific, Millville, N.J.). The flaskswere attached to a gas manifold by hypodermic needles andfurther deoxygenated by five rounds of alternate evacuationand introduction of N2 finishing with an N2 overpressure.Cells (500 ,ul) were injected and incubated for 1 h to exhaustthe remaining 02. The substrate was then injected to give afinal concentration of 3.2 mM. An aerobic control was set upby using the same cell volumes and reagent concentrationsas in the anaerobic treatment. Both treatments were thenplaced on a shaker, and one flask was removed at each timepoint and acidified with 50 ,lI of 10 N H2SO4 to stop cellactivity.DCBa cometabolism. Cells were grown in MSM containing

8.2 mM benzoate and 3.2 mM DCBa. Samples (1 ml) wereremoved daily for absorbance and inorganic chloride deter-minations. After 10 days of incubation, fresh benzoate was

supplied to the cells by the addition of 0.5 ml of sterile 40mM benzoate solution.

Metabolite recovery. Samples, which had previously beenacidified, were brought to 50 ml with distilled water andextracted twice with an equal volume of ethyl acetate. Theorganic phases were pooled, dried over Na2SO4, and evap-orated with a Buchi rotary evaporating unit (BrinkmannInstruments, Westbury, N.Y.). The dried material wastransferred from the Rotovap flasks in 1 ml of methanol thatwas then evaporated under a stream of N2. The sample was

finally dissolved in 10 to 100 ,ul of methanol.Preparation of cell extracts. The cell suspension was pre-

pared in the same manner as described above for aerobicresting cell incubations. Cells were disrupted by passagethrough a French pressure vessel operated at 25 MPa. Celldebris was then removed by centrifugation at 48,000 x g for

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2.0

-JE0

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FIG. 1. Growth curves of P. aeruginosa JB2 on mono-CBas, DCBas, and TCBa.

45 min. Cell extracts were supplemented with 0.2 mMNADH and 0.1 mM flavin adenine dinucleotide (1) in mono-oxygenase assays.

Plasmid DNA preparation. The following procedures wereused for plasmid DNA isolation: alkaline lysis (26), polyeth-ylene glycol precipitation (16), boiling lysis (19), and directlysis (7). DNA isolated by these procedures was separated in0.7% agarose minigels (26) at 50 V for 2 h at 4°C.Mutant isolation. Spontaneous mutants of JB2 were ob-

tained by inoculating single colonies grown on 2-CBa intoliquid MSM (1% glycerol). After 2 days, the glycerol cultureswere diluted and plated on MSM (1% glycerol). Singleglycerol-grown colonies were then picked and transferred tothe master plate (MSM with 1% glycerol). After 2 days ofgrowth, colonies were transferred from the master plate toMSM plates containing benzoate, 2-CBa, 3-CBa, 2,3-DCBa,or 2,5-DCBa. Growth was then scored after 10 days ofincubation.Oxygen uptake measurements. Cells were prepared for

oxygen uptake experiments as described above for aerobicresting cells. Measurements were made with an oxygenelectrode (Yellow Springs Instrument Co., Yellow Springs,Ohio) in a 2-ml cell at 30°C. All substrates (10 ,ul) were addedfrom 10-mg-ml-1 methanol stocks except 3-chlorocatechol,which was 6.5 mg ml-'. All rates were corrected for endog-enous uptake. No oxygen consumption was observed inresponse to the addition of methanol alone.

RESULTSStrain JB2 readily used 2-CBa, 3-CBa, and 2,5-DCBa as

growth substrates with doubling times of 3.3, 9.9, and 9.3 h,

respectively. In contrast, growth on 2,3-DCBa was muchslower (td = 31 h), although all growth substrates supportedroughly the same maximum cell density (Fig. 1). Strain JB2also used 2,3,5-TCBa (td = 14.2 h) as a sole carbon andenergy source (Fig. 1) with complete stoichiometric chloriderelease (data not shown).

In addition to the CBas listed above, strain JB2 used awide range of substituted benzoic acids as sole carbonsources including 2-bromo-, 2,5-dibromo-, 2-iodo-, 2-fluoro-,4-fluoro-, 2-hydroxy-, 2,3-dihydroxy-, 2,5-dihydroxy-, 3,4-dihydroxy-, and 2-hydroxy-5-chloro-. The following substi-tuted benzoates did not serve as growth substrates for strainJB2: 4-chloro-, 2,4-dichloro-, 2,6-dichloro-, 3,4-dichloro-,3,5-dichloro-, 3-bromo-, 4-bromo-, 3-iodo-, 4-iodo-, 3-flu-oro-, 2,4-dihydroxy-, 2,6-dihydroxy-, 2-hydroxy-3-chloro-,and 2-hydroxy-4-chloro-. Whereas phenol did serve as agrowth substrate for strain JB2, no growth or chloriderelease occurred with monochlorophenols or the dichlo-rophenols examined.DCBas not used as growth substrates by strain JB2 were,

to varying degrees, cometabolically dechlorinated. After 10days of growth on benzoate (8.2 mM) in the presence of2,4-DCBa (3.2 mM), chloride release totaled 1.2 mM. Incontrast, chloride release in the presence of 2,6-, 3,4-, and3,5-DCBa was 0.7, 0.3, and 0.6 mM, respectively. Theextent of 2,4-DCBa cometabolism was dependent on theamount of growth substrate present: supplying cells withadditional benzoate initiated further degradation of 2,4-DCBa (Fig. 2). GC analysis of the growing cell cultures thatcometabolized 2,4-DCBa revealed only the residual sub-strates; no intermediates could be detected.

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2.9

1.9

ioolt2E0

0

0.9

-0.10 5 10 15 20

dayFIG. 2. Chloride release from 2,4-DCBa by strain JB2 while grown on benzoate (Ba). Arrow refers to the point when cultures were

respiked with benzoate.

Mono-CBas or DCBas were degraded in aerobic, but notanaerobic, resting cell incubations. Four intermediates wereidentified from the degradation of the mono-CBa and DCBa(Fig. 3) by aerobic resting cells. Since strain JB2 did notgrow on or metabolize mono- or dichlorophenols, theseproducts were most likely produced by the acid-catalyzeddehydration of the corresponding dihydrodiols. Dehydrationof dihydrodiols under acidic conditions has been reportedpreviously (14). Analysis of the CBa stock solutions byGC-mass spectrometry indicated that the starting materialswere not contaminated with mono- or dichlorophenols.Mono-CBas and DCBas were also tested for the ability to

elicit monooxygenase activity in cell extracts. Relativelyhigh oxygen uptake rates were observed in response tocatechol, 3-chlorocatechol, and 4-chlorocatechol (68, 74,and 42 nmol of 02 min-1, respectively). Activity was notelicited by mono-CBas or DCBas in the cell extracts with orwithout NADH and flavin adenine dinucleotide supple-ments.Benzoate oxidation rates were diminished in CBa-grown

cells relative to benzoate-grown cells (Table 1). In contrast,growth on CBas resulted in increased relative activity levelson CBas. The exceptions to this trend were 2,3-DCBa- and2,5-DCBa-grown cells, which did not respond to additions of3-CBa. CBa-grown cells were also induced for the oxidationof chlorocatechols (Table 1). This was most pronounced in2,5-DCBa-grown cells in response to both 3- and 4-chloro-catechol.As a first step toward investigating the genetic organiza-

tion of CBa degradation, a search was undertaken to identify

plasmid DNA. Four protocols (7, 16, 19, 26) were used, noneof which yielded plasmid DNA. In most cases, a single bandmigrating the same distance as linear DNA (i.e., the approx-imate migration distance of the 23.1-kb fragment of HindIll-digested lamda DNA) was observed on gels. Digestion ofthese samples with endonuclease HindIII or EcoRI resultedin smearing rather than discrete fragmentation. Thus, theDNA isolated was linear and not covalently closed circular.As positive controls for the effectiveness of the alkaline lysismethod (26), three defined strains (24), P. putida R5-3, P.putida CB1-9, and P. alcaligenes C-0, known to harborplasmids ranging in size from 33 to 57 kb were examinedalong with strain JB2. Plasmid DNA was isolated from eachcontrol but not from strain JB2.To aid in the location of the CBa degradation genes,

mutants were sought. Mutagenesis with TnS, TnWO, or Tn9was unproductive as strain JB2 was intrinsically resistant tokanamycin, tetracycline, and chloramphenicol at levels up to500, 100, and 200 ,ug ml-1, respectively. Mutants werefinally obtained when it was observed that subculturing ofstrain JB2 on glycerol resulted in a very high frequency ofspontaneous 2CBa- mutants. In a survey of 100 glycerol-grown colonies, 70% were 2-CBa-. Examination of themutants showed that the loss of 2-CBa activity coincidedwith the loss of 2-halobenzoate utilization in general (includ-ing 2,3-DCBa and 2,5-DCBa), yet the mutants retained theability to utilize 3-CBa and 3-bromobenzoate.The oxygen uptake rates of the mutants were examined to

identify the step in the degradation pathways at which theblockage occurred (Table 2). Relative to the wild type,

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3846 HICKEY AND FOCHT

100-

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FIG. 3. Mass spectra of products identified from strain JB2 resting cell incubations (substrate in parentheses). (A) 3-Chlorocatechol(2-CBa); (B) 3-chlorophenol (3-CBa); (C) 4-chlorocatechol (2,3- and 2,5-DCBa); (D) 2,3-dichlorophenol (2,3-DCBa).

mutant cells displayed reduced oxidation rates on all CBaswith the exception of 3-CBa. Moreover, oxidation ofhalobenzoates in general was reduced in the mutants. Theactivity on 3-chlorocatechol, however, was found to besimilar with both cell types.

DISCUSSION

P. aeruginosa JB2 utilized a range of halobenzoates thathas not been described previously. Strain JB2 was also, tothe best of our knowledge, the first organism reported to use

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HALOBENZOATE DEGRADATION IN P. AERUGINOSA JB2

80

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hourFIG. 4. Aerobic versus anaerobic formation of 4-chlorocatechol from 2,5-DCBa (2.6 mM) by 2,5-DCBa-grown resting cells of strain JB2.

a TCBa as a growth substrate. The possibility was consid-ered that, by using 2-CBa as an enrichment substrate,organisms with unusually versatile degradative capabilitiescan be selected. This, however, appears not to be the case,as other organisms isolated on 2-CBa (2, 8, 9, 33, 35, 40)have not been reported to utilize a similar range of mono-,di-, or trihalobenzoates.

Degradation of both 2- and 3-CBas is proposed to proceedthrough 3-chlorocatechol (Fig. 5). 3-Chlorocatechol has not

TABLE 1. Oxygen uptake by strain JB2 whole cells grownon the indicated substrate

Test % Oxygen uptake with given growth substrateasubstrate Benzoate 2-CBa 3-CBa 2,3-DCBa 2,5-DCBa

Catechol 100 (373) 100 (294) 100 (178) 100 (84) 100 (117)3-Chlorocate- 7 15 13 7 107

chol4-Chlorocate- 17 24 39 52 93

cholBenzoate 100 (469) 100 (117) 100 (128) 100 (43) 100 (26)2-CBa 20 71 34 100 453-CBa 18 24 34 ND ND2,3-DCBa 5 15 3 100 202,5-DCBa 16 77 34 198 323

Values in parentheses are oxygen uptake rates (nanomoles of 02 perminute per milligram of protein) measured on benzoate and catechol. Ratesare expressed as a percentage of that measured on benzoate (for benzoate andchlorobenzoate determinations) or catechol (for catechol and chlorocatecholdeterminations). ND, No measurable difference from basal respiration rate.

previously been identified from 2-CBa, but was postulatedby Sylvestre et al. (35) based on the detection of chloromu-conic acid. These investigators also identified catechol as a

product of 2-CBa degradation as have others (8, 9, 17, 40).Furthermore, the formation of catechol from 2-CBa has beendemonstrated, at least in one case (10), to be dioxygenasemediated. The main mechanism for 2-CBa degradation thenappears to be dioxygenation, resulting in the formation ofeither catechol or, as in the case of strain JB2, 3-chlorocat-echol.The lack of anaerobic transformation of DCBas eliminated

TABLE 2. Comparison of oxygen uptake by wild-type andmutant cells of strain JB2 grown overnight on benzoate (3.2 mM)

Rate (%)aSubstrate

Wild type Mutant

Benzoate 100 (311) 100 (188)2-CBa 30 93-CBa 16 262,3-DCBa 12 42,5-DCBa 12 42-Bromobenzoate 22 22-Iodobenzoate 28 72-Fluorobenzoate 40 173-Bromobenzoate 20 15Catechol 100 (373) 100 (212)3-Chlorocatechol 7 74-Chlorocatechol 17 38

a See footnote a, Table 1.

* anaerobicX aerobic

-- 1.

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3848 HICKEY AND FOCHT

I~~I

CO2H

I I

Xci

CO2H

III -CI

CO2H

IlU l

CI -OH

ring rcleauage

ringcleauage

CI

FIG. 5. Proposed degradation pathways of mono-CBAs and DCBas by strain JB2. I, 2-CBa; II, 3-CBa; III, 2,3-DCBa; IV, 2,5-DCBa.

reductive dechlorination, such as that described for 2,4-DCBa-degrading organisms (37, 41), and hydrolytic dechlo-rination, similar to that of 4-CBa degraders (1, 23, 27, 37), aspossible degradative mechanisms. The initial reactions in the2,3- and 2,5-DCBa degradative pathways were thereforemediated by either mono- or dioxygenases. Of the two,dioxygenase involvement was more likely for the followingreasons. First, CBas failed to elicit NADH-dependent mono-oxygenase activity in cell extracts. Second, 2,3-dichlorophe-nol, identified from the degradation of 2,3-DCBa, would beproduced during extraction by the acid hydrolysis of 2,3-dichloro- 1- carboxy-1,6- dihydrodiolcyclohexadiene. Thisdichlorodihydrodiol would be a primary intermediate pro-duced from dioxygenation of 2,3-DCBa. 2,3-Dichlorophenolwas unlikely to be a metabolite as strain JB2 did not grow onor metabolize mono- or dichlorophenols (data not shown).Third, formation of 4-chlorocatechol from 2,5-DCBa couldresult from dioxygenation at the 1,2 positions. This type ofdioxygenation would spontaneously remove a chloride fromthe ortho-carbon atom that bears the OH group. A similarmechanism has been proposed for formation of catecholfrom 2-CBa as discussed above.

In previous reports (18, 29), CBa degradation was consid-ered to be limited by a relatively narrow-spectrum benzoate-

dioxygenase. In these cases, utilization of CBas elicitedhyperexpression of the benzoate dioxygenase to compensatefor the low affinity of the enzyme for the chlorinated sub-strate. The findings of Knackmuss and co-workers (6, 18, 29)indicated that a broad-spectrum dioxygenase was necessaryfor efficient use of a range of CBas. Data from oxygen uptakeexperiments, however, indicated that strain JB2 circum-vented the limitations of the benzoate dioxygenase by induc-ing a second dioxygenase with greater halobenzoate speci-ficity. A similar situation has been reported with P. putidaCLB 250 (8), in which growth on 2-CBa led to increasedactivity on 2-halobenzoates without a concomitant increasein activity on benzoate. Based on these findings, strain CLB250 was postulated to possess a separate halobenzoatedioxygenase.

Further evidence for a separate halobenzoate dioxygenasein strain JB2 was provided by studies with the spontaneousmutants. The mutants clearly retained benzoate dioxygenaseactivity as growth and oxygen uptake activity on benzoatewere unaffected. The mutants also retained wild-type activ-ity on chlorocatechols and thus were not disabled in CBadegradation due to a blockage at this stage in the pathway.The capacity of the mutants to oxidize CBas, however, wasobviously diminished. The observations outlined above,

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HALOBENZOATE DEGRADATION IN P. AERUGINOSA JB2

together with the evidence for dioxygenase involvement inCBa degradation, indicated that the mutant phenotype wasmost likely attributable to the loss of a halobenzoate dioxy-genase.Although plasmid DNA was not isolated by any of the

procedures used, the high frequency of spontaneous mutantsmight be taken as evidence that the genes were carried on anundetected plasmid. Plasmid-borne 2-CBa degradation geneswere also implicated in another strain of P. aeruginosadesignated B16 (33). Similar to P. aeruginosa JB2, strain B16also gave rise to spontaneous 2-CBa- mutants, albeit at alower frequency (3% in nutrient broth-grown cells). Thissimilarity between strains JB2 and B16 suggested a commongenetic basis, such as a plasmid, for CBa degradation inthese organisms.

In conclusion, the work described above with P. aerugi-nosa JB2 elucidated novel bacterial capabilities for haloben-zoate degradation vis-a-vis substrate range and the potentialinvolvement of halobenzoate dioxygenases. Evidence for ahalobenzoate dioxygenase in strain JB2 was consistent withfindings from other workers, suggesting that "specialized"dioxygenases may represent an alternate approach to CBaoxidation.

ACKNOWLEDGMENTSThis work was supported in part by grants from the University of

California Biotechnology Research and Education Program and theOccidental Chemical Corp.

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