JOURNAL OF BACTERIOLOGY, Mar. 1989, p. 1428-14340021-9193/89/031428-07$02.00/0Copyright X) 1989, American Society for Microbiology

Vol. 171, No. 3

Microbial Degradation of 1-Chlorinated Four-CarbonAliphatic Acids

DORIS KOHLER-STAUB AND H.-P. E. KOHLERt*

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

Received 3 June 1988/Accepted 24 November 1988

Alcaligenes sp. strain CC1 is able to grow on several a-chlorinated aliphatic acids (2-chlorobutyrate,2-chloropropionate, and chloroacetate), as wel as on the n-chlorinated four-carbon aliphatic acids trans-

3-chlorocrotonate, cis-3-chlorocrotonate, and 3-chlorobutyrate as sole carbon and energy sources. Dehaloge-nation of a-chlorinated acids could be measured by using resting cells grown on all the different carbon sources,whereas dehalogenation of P-chlorinated four-carbon acids could be detected only by using resting ceUls grownon four-carbon compounds. A constitutive 2-haloacid dehalogenase, which did not show any activity with,8-chlorinated four-arbon acids, was detected in cell extracts. Cell extracts of crotonate-grown cellsadditionally contained a 1-haloacid dechlorination activity, which acted on trans-3-chlorocrotonate, cis-3-chlorocrotonate, and 3-chlorobutyrate and was strictly dependent on coenzyme A, ATP, and Mge.Dechlorination of 13-chlorinated four-carbon acids takes place after activation of the acids to their coenzyme Aderivatives and seems to be independent of the constitutive 2-haloacid dehalogenase.

Halogenated aliphatic compounds, which are in use assolvents, pesticides, and starting materials for chemicalsynthesis, are introduced into the environment in largequantities by direct application or by inadequate wastedisposal by both producers and consumers (23). Chlorinationof drinking water is another process that results in theformation and distribution of these xenobiotic chemicals(19).

Microbial degradation of halogenated aliphatic com-pounds has been studied to an increasing extent within thelast three decades. Although the more recent reports mainlydescribed aerobic and anaerobic bacterial degradation ofvarious haloalkanes, research in this field originally startedout with studies on the mineralization of halogenated al-kanoic acids (15, 18). Except for an early study by Bollagand Alexander (3), which deals with bacterial dehalogena-tion of t-chlorinated aliphatic acids, all investigations con-cerning bacterial degradation of haloacids have focused onsaturated a-halogenated acids (14, 20, 27). Two differenttypes of enzymes, haloacetate dehalogenase (EC 3.8.1.3)and 2-haloacid dehalogenase (EC 3.8.1.2), that specificallycatalyze the hydrolytic removal of halosubstitutes on thea-carbon of aliphatic acids have been described (28).Some halogenated aliphatic compounds may be produced

in soil, sediments, or sewage sludge by indigenous microor-ganisms in the course of biotransformation reactions ofhalogenated pollutants that originally had a more complexchemical structure. The fate of chlorinated aliphatic degra-dation products is not generally a major topic of study, sincetheir biodegradation is assumed to be nonproblematical.Nevertheless, it is well established (1, 31) that chlorinatedaliphatic dead-end products are produced from the nonspe-cific microbial attack of chlorinated aromatic hydrocarbonsthrough the meta-pyrocatechase pathway. Microbial trans-formations of polychlorinated biphenyls (PCBs) lead tochlorobenzoates after meta-cleavage of one of the rings andsubsequent hydrolysis of the aliphatic side chain (10); al-

* Corresponding author.t Present address: EAWAG, Abteilung fur Technische Biologie,

Ueberlandstrasse 133, CH-8600 Dubendorf, Switzerland.

though never isolated or identified, the second reactionproduct is expected to be a chlorinated aliphatic five-carbonacid (32), as based on the original discovery of the metafission pathway by Dagley et al. (6). Nevertheless, theseproducts ofPCB degradation do not appear to be refractile tomicrobial attack by indigenous soil microorganisms (5, 9).Unfortunately, the microorganisms which metabolize thedegradation products of PCBs cannot be isolated, becausethe compounds are not commercially available.

Since Acinetobacter sp. strain P6, a well-studied PCBcometabolizing strain (11, 21), grows on 3-chlorobutyric acidbut not on the cis- and trans-isomers of 3-chlorocrotonic acid(3-chlorobutenoic acids), we chose these compounds forisolation of microorganisms that might have the capacity tometabolize the aliphatic end products ofPCB cometabolism.Moreover, chlorocrotonic acid is the only unsaturated 1B-chloroaliphatic acid that is commercially available. In thiscommunication we report bacterial growth on cis- andtrans-chlorocrotonic acid as well as on 3-chlorobutyric acidand propose a pathway for their degradation.

MATERLILS AND METHODSMedia and growth conditions. The minimal salts medium

(MM) we used consisted of 20 mM phosphate buffer(KH2PO4, Na2HPO4 [pH = 7.2]), 0.5 g of (NH4)2S04, 0.1 gof MgSO4- 7H20, and 0.075 g of Ca(NO3)2 in deionizedwater (1.0 liter) supplemented at 1 mI/liter with a traceelement stock solution containing (in grams per liter) thefollowing: FeSO4 7H20, 1; MnSO4. H20, 1; Na2MoO4-2H20, 0.25; H3B03, 0.1; CuCl2- 2H20, 0.25; ZnCl2, 0.25;NH4VO3, 0.1; Co(NO3)2 - 6H20, 0.25; NiSO4 * 6H20, 0.1.Carbon sources were added at 5 mM; acids were neutralizedwith an equimolar amount of NaOH. Luria broth (24) wasused for preparing complex liquid or solid medium. Cellswere grown at 28°C on a rotary shaking platform (150 rpm).

Analytical procedures. (i) Chlorocrotonic acids. The twoisomers of chlorocrotonic acid were analyzed by gas chro-matography after derivatization to their correspondingmethyl esters. Culture supernatants were obtained by cen-trifugation at 14,000 x g for 3 min. A 10.pl portion wasremoved, mixed with 200 pl of BF3-methanol (Supelco, Inc.,

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MICROBIAL DEGRADATION OF ALIPHATIC ACIDS 1429

Bellefonte, Pa.), and heated to 60°C for 30 min. Afteraddition of 500 ,ul of 15% sodium chloride solution, methylesters were extracted with 5 ml of hexane and subsequentlyinjected (1 p.l) into an HP 5890A gas chromatograph(Hewlett-Packard Co., Palo Alto, Calif.) equipped with a63Ni electron capture detector, a DB5 capillary column (30 mby 0.25 mm [inner diameter]; film thickness, 0.25 ,Lm; J&WScientific, Folsom, Calif.), and a data system (Nelson Ana-lytical Inc., Cupertino, Calif.). The carrier gas was heliumwith a linear flow velocity of 30 cm/s at 100°C. Analysis wascarried out under isothermal conditions at 90°C. Retentiontimes were 4.1 and 2.9 min for the ester derivatives of thetrans and cis isomers of 3-chlorocrotonic acid, respectively.Since no standards of the methyl esters of the chlorocrotonicacids were commercially available, the identity of our de-rivatization products was confirmed by gas chromatography-mass spectrometry (HP 5970 Series mass selective detector;Hewlett-Packard Co.). Both methyl esters resulted in iden-tical mass spectrometry patterns containing the followingfragments: 136 (33%) (M + 2)+; 135 (6%) (M + 1)+; 134(100%) (M)+; 105 (105%) (M + 2 - 31)+, loss of O-CH3;104 (16%) (M + 1 - 31)+, loss of -O-CH3; 103 (306%) (M- 31)+, loss of----CH3; 100 (6%) (M + 1 - 35)+, loss ofchloride; 99 (108%) (M + 2 - 37)+ and (M - 35)+, lossof chloride; 77 (17%) (M + 2 - 31 - 28)+, loss of-CO---O--CH3; 75 (53%) (M - 31 - 28)+, loss of-CO--O--CH3; 67 (20%); 59 (33%), -CO--O-CH3 frag-ment; 49 (27%); 39 (180%), C3H3 fragment.

(ii) Chloride. The chloride concentration in culture fluids,in resting-cell assays, and in assays with cell extracts wasdetermined spectrophotometrically at 460 nm with mercuricthiocyanate and ferric nitrate as described by Bergmann andSanik (2). Prior to the analysis, the samples were freed ofprotein and cells by acid precipitation and centrifugation.Samples containing coenzyme A (CoA), which interferedwith the chloride measurement, were treated with 50%(vol/vol) hydrogen peroxide (30%) and then heated at 70°Cfor 30 min.

(iii) Protein determination. The protein content in cellsuspensions and in cell extracts was measured by the biuretmethod (12) and the method of Bradford (4), respectively,with bovine serum albumin as the standard.

Preparation of washed cell suspensions and cell extracts.Cells were harvested from the late exponential growthphase, centrifuged for 15 min at 12,000 x g, suspended in 20mM phosphate buffer (pH 7.2), centrifuged again, and finallyresuspended in phosphate buffer (pH 7.2; 5 mUg of wetcells). Cell breakage was performed by two passages througha French press at 20,000 lb/in2. Clarified cell extracts wereobtained by centrifugation for 20 min at 40,000 x g.

2-Haloacid dehalogenase assay. To determine the 2-halo-acid dehalogenase activity in cell extracts, a reaction mix-ture (1 ml) containing 50 mM Tris-H2SO4 (pH 8.0) and 2.5mM chlorinated compound was incubated at 30°C. Theenzyme reaction was started by adding cell extract to give0.3 mg of protein per ml. Samples of 200 ,ul were takenperiodically and analyzed for chloride. Spontaneous dechlo-rination was determined for each substrate and used forcorrection of enzymatic dechlorination rates. Specific deha-logenase activity (in milliunits) was expressed as nanomolesof chloride per minute per milligram of protein.

II-Hydroxyacyl-CoA dehydrogenase assay. The ,-hydroxy-acyl-CoA dehydrogenase activity in cell extracts of croto-nate-grown cells ofAlcaligenes sp. strain CC1 was measuredby monitoring NADH formation at 340 nm (Uvikon 860;Kontron Instruments Inc., Everett, Mass.). The assay mix-

ture contained 50 mM phosphate buffer (pH 7.5), 1 mMfour-carbon acid, 1 mM CoA, 1 mM ATP, 1 mM Mg2+, and1.5 mg of protein per ml. Rates are expressed as nanomolesof NADH formed per minute per milligram of protein(milliunits).

Assay for dechlorination of n-chlorinated haloacids. Thedechlorination of ,B-chlorinated haloacids in cell extracts ofcrotonate-grown Alcaligenes sp. strain CC1 was measuredby monitoring substrate disappearance and chloride releaseduring or after incubation at 30°C. The reaction mixturecontained 50 mM phosphate buffer (pH 7.5), 3 mg of proteinper ml, 1 mM substrate, 2 mM CoA, 2 mM ATP, and 1 mMMg2+.

Chloride release by resting cells. Resting-cell suspensionsof Alcaligenes sp. strain CC1 grown on different chlorinatedand unchlorinated compounds were incubated in 50 mMTri-H2SO4 buffer (pH 8.0) containing 2.5 mM of the chlori-nated substrate (0.5 mg of protein per ml). Specific rateswere expressed as nanomoles of chloride released perminute per milligram of protein.

Chemicals. trans-3-Chlorocrotonic acid, cis-3-chlorocro-tonic acid (95% purity; 5% cross-contamination with theother isomer, respectively), and 3-chlorobutyric acid wereobtained from Dixon Fine Chemicals, Sherwood Park, Al-berta, Canada. 4-Chlorobutyric acid and 2-chlorobutyricacid were purchased from Fluka, Buchs, Switzerland. Allother chlorinated and unchlorinated aliphatic acids werefrom Aldrich Chemical Co., Inc., Milwaukee, Wis. CoA,NAD, and ATP were purchased from Sigma Chemical Co.,St. Louis, Mo.

RESULTS

Isolation and properties of Alcaligenes sp. strain CC1. Toscreen for microorganisms able to grow on chlorocrotonicacid, we inoculated minimal medium (50 ml) containing 5mM trans-3-chlorocrotonic acid as the sole carbon sourcewith activated sewage taken from the Riverside MunicipalSewage Treatment Plant, Riverside, Calif. After repeatedtransfers to selective liquid and solid media, single coloniescapable of growing on trans-3-chlorocrotonic acid wereisolated. The isolate was an obligately aerobic, gram-nega-tive, nonmotile coccoid rod, which was both oxidase andcatalase positive. Carbohydrate metabolism was nonfermen-tative, and denitrification could not be observed. Besidesseveral chlorinated aliphatic acids, the isolate utilized ben-zoate, crotonate, butyrate, 1-hydroxybutyrate, propionate,and acetate. According to taxonomic descriptions (37), theorganism was designated as Alcaligenes sp. strain CC1. Ithas been deposited with the American Type Culture Collec-tion as ATCC 49033.Growth characteristics. Growth of Alcaligenes sp. strain

CC1 on the two isomers of chlorocrotonic acid resulted inthe disappearance of the substrates and the simultaneousformation of biomass and equimolar amounts of chloride(Fig. 1).

Several additional chlorinated alkanoic acids were utilizedby CC1 as the sole carbon sources (Table 1). These chemi-cals can be divided into two groups: one group comprisesa-chlorinated compounds, and the other group comprises1B-chlorinated four-carbon acids. 3-Chloropropionate, a 13-chlorinated three-carbon acid, did not serve as a growthsubstrate. Spontaneous dechlorination was assessed for allchlorinated growth substrates to exclude the possibility ofgrowth on the dechlorinated acids (Table 1). 3-Chlorobu-tyrate was dechlorinated spontaneously in sterile neutral

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1430 KOHLER-STAUB AND KOHLER

.0'

0.8

0.6

0.4

0

> 0.2

cnz

w ol-J

2 1.0A0.0

0.8

0.6

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0l

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4 4u

3-^

02 a:0

I

0

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zw

0

-J

U

0I0o

0

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HOURS

FIG. 1. Growth of Alcaligenes sp. strain CC1 on cis-3-chlorocro-tonate (CCC) and trans-3-chlorocrotonate (TCC). (A) Growth on

TCC. Symbols: A, TCC (mM); 0, A546; 0, chloride (mM). (B)Growth on CCC. Symbols: A, CCC (mM); 0, A546; 0, chloride(mM).

medium at a low rate, but the chloride release rate was

significantly enhanced during bacterial growth as a result ofan enzymatic dechlorination mechanism (Fig. 2). Moreover,the only -y-chloroalkanoic acid tested, 4-chlorobutyrate, de-composed chemically in sterile medium, with the release ofall the chloride in 3 h, and thus was an unsuitable substrate,for assessing microbial dechlorination. In contrast, trans-3-chlorocrotonic acid and cis-3-chlorocrotonic acid werevery stable in regard to spontaneous chemical dechlorinationunder physiological conditions.Growth yields of Alcaligenes sp. strain CC1 did not differ

greatly among the substrates tested (Table 1). Degradation ofthe chlorinated acids neither required nor produced anyadditional energy beyond that incurred in the degradation ofthe nonchlorinated acids.

0

4

E

3 LLJ

0c]0

2 JI

l0 20 30

HOURS

FIG. 2. Growth of Alcaligenes sp. strain CCM on 3-chlorobu-tyrate. Symbols: 0, A546; 0, chloride in inoculated sample (mM);A, chloride in sterile control (mM).

Dechlorination of ot-chlorinated acids with resting cells andcell extracts. Resting cells of Alcaligenes sp. strain CC1grown on various chlorinated and unchlorinated carbonsources dechlorinated all (x-chlorinated aliphatic acids tested(Table 2). Moreover, a constitutive 2-haloacid dehalogenaseacting on (x-chloroalkanoic acids could be detected in cellextracts of Alcaligenes sp. strain CC1 (Table 3). The pHoptimum was found to be 9.0. The enzyme did not requireany additional cofactors for activity. 3-Chlorinated acidswere not substrates of the 2-haloacid dehalogenase, sincechloride was not released from them.

,B-Hydroxyacyl-CoA dehydrogenase activity. The mostlikely pathway for catabolism of four-carbon fatty acids bymicroorganisms is n-oxidation. In this sequence, the four-carbon fatty acid is activated to its CoA thioester in an ATP-and CoA-dependent reaction, which is followed by hydra-tion and NAD-dependent oxidation of the P-carbon cata-lyzed by the P-hydroxyacyl-CoA dehydrogenase.NADH production was observed from crotonate and

3-chlorobutyrate when incubated with cell extracts of croto-nate-grown cells. The reaction required ATP, CoA, andMg2+ (Table 4). In contrast, NAD-dependent oxidation ofP-hydroxybutyrate did not involve activation by CoA (Table4). This is in accordance with the degradation of the energystorage compound poly-p-hydroxybutyrate; after depoly-merization, 3-hydroxybutyrate is oxidized to acetoacetate

TABLE 1. Utilization of chlorinated and unchlorinated aliphatic acids by Alcaligenes sp. strain CCl

Yield (mg of Chloride production (mM)b in:Growth substratea Generation protein/Cmolo Inocuated Steriletime (h) substrate C) mncuaediu coterole

trans-3-Chlorocrotonate 3.7 6.0 5.3 <0.02cis-3-Chlorocrotonate 3.3 6.0 4.9 0.023-Chlorobutyrate 4.6 5.5 4.9 0.402-Chlorobutyrate 15.0 4.8 2.7 0.032-Chloropropionate 6.3 5.3 4.0 0.14Chloroacetate 6.3 ND' 5.5 0.02Crotonate 5.1 6.8Butyrate 5.1 5.5P-Hydroxybutyrate ND 6.5Propionate ND 6.3"

No growth was observed with 3-chloropropionate, dichloroacetate, trichloroacetate, and 2,2-dichloropropionate.b The minimal medium used contained 5 mM of the chlorinated substrates.' ND, Not determined.

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MICROBIAL DEGRADATION OF ALIPHATIC ACIDS 1431

TABLE 2. Dechlorination activity of resting cells of Alcaligenes sp. strain CCM after growth on various carbon sources

Specific chloride release rate from':

Growth substrate trans-3- cis-3-Chloro- Chloro- 3-Chloro- 2-Chloro- 2,2-Dichloro- Chloro- Dichloro-

crotonate crotonate butyrate propionate propionate acetate acetate

trans-3-Chlorocrotonate 11 11 11 235 22 531 184cis-3-Chlorocrotonate 5 5 5 232 8 412 173Crotonate 10 28 57 110 52 105 52,B-Hydroxybutyrate 9 12 29 104 12 186 942-Chloropropionate <2 <2 <2 78 16 88 85Propionate <2 <2 <2 64 5 108 60Chloroacetate 0 0 0 102 19 165 97Acetate 0 0 0 99 9 169 103Benzoate 0 0 0 83 22 204 168

a Dechlorination rates were calculated on the basis of nanomoles of chloride per minute per milligram of protein (milliunits).

by an NAD-specific ,3-hydroxybutyrate dehydrogenase (13).With trans-3-chlorocrotonate and cis-3-chlorocrotonate,whose p-carbon is in the same oxidation state as the one of,-ketobutyrate (i.e., +2), no NADH formation could beobserved (Table 4). However, both compounds showed astrong inhibitory effect on NADH formation when added toassays with crotonate as a substrate; this would indicateinterference with an earlier reaction of the p-oxidationsequence.

Dechlorination of Is-chlorinated four-carbon acids with rest-ing cells and cell extracts. Dechlorination of P-substitutedfour-carbon acids occurred only with resting cells of Alcali-genes sp. strain CC1 grown on four-carbon compounds(Table 2). This indicates that p-dechlorination is inducible, incontrast to a-dechlorination, which is constitutive.

Cell extracts of crotonate-grown Alcaligenes sp. strainCC1 contained a dechlorination activity, which acted ontrans-3-chlorocrotonate, cis-3-chlorocrotonate, and 3-chlo-robutyrate and was strictly dependent on the presence ofCoA, ATP, and Mg2+ (Fig. 3; Table 5). This shows that thedechlorination step occurs after an ATP-requiring activationof trans-3-chlorocrotonate, cis-3-chlorocrotonate, and 3-chlorobutyrate with CoA. Addition ofNAD did not have anyeffect on chloride release from 3-chlorobutyrate (Table 5).This compound apparently is dechlorinated while still beingat an oxidation level lower than the one of the chlorocroto-nyl-CoA derivates.

DISCUSSIONWe have provided evidence that the dechlorination of

p-chlorinated four-carbon fatty acids by Alcaligenes sp.

strain CC1 is dependent on a prior reaction of the aliphaticacids with CoA, which presumably results in the formationof the corresponding CoA esters. Although we could notisolate any intermediates, we assume that this esterificationis followed by the removal of the chlorine substitute, allow-ing the four-carbon acids to be further catabolized accordingto the scheme of p-oxidation (Fig. 4). This degradationpathway represents another case in which the dechlorinationreaction is not the initial catabolic event. According to thenumerous studies on microbial degradation of halogenatedcompounds, both strategies-initial dehalogenation and de-halogenation in a later stage of substrate catabolism-exist.Anaerobic degradation sequences of halogenated aromatics(7, 36) as well as aliphatic compounds (8) are always initiatedby a reductive dehalogenation reaction. Both strategies havebeen observed for aerobic degradation of haloaromatics:Pseudomonas sp. strain CBS3 and an Arthrobacter sp. startthe degradation of 4-chlorobenzoate with a dechlorinationstep (25, 29), whereas mutants derived from Pseudomonassp. strain B13 metabolize 3-chlorobenzoate and 4-chloroben-zoate to chlorinated cis-,cis-muconates, which are subse-quently subjected to enzymatic dechlorination (30). Amongthe degradation pathways of haloaliphatic compounds, aninitial removal of the chlorine substitutes seems to becommon, as reported for all 2-haloalkanoic acids (26, 38),dichloromethane (22, 34), and monohaloalkanes (33). Incontrast, chloroethanol is first oxidized to chloroacetic acid,which is then dechlorinated by a conventional 2-haloaciddehalogenase (16, 35). Degradation of biterminally haloge-nated alkanes can involve both strategies, as shown for1,2-dichloroethane by Janssen et al. (17). After hydrolytic

TABLE 3. 2-Haloacid dehalogenase activity in cell extracts of Alcaligenes sp. strain CCM grown on different carbon sources

Specific dehalogenase rate for":

Growth substrate trans-3- cis-3-Chloro- Chloro- 3-Chloro- 2-Chloro- 2,2-Dchloro- Chloro- Dichloro-crotonatecrotonate butyrate propionate propionate acetate acetatecrotonate crotonate

trans-3-Chlorocrotonate 0 0 0 977 35 1,319 183cis-3-Chlorocrotonate NDb ND ND 1,335 34 1,242 226Chloroacetate ND ND ND 1,059 114 1,415 3272-Chloropropionate 0 0 0 1,427 300 1,632 599Crotonate 0 0 0 1,086 113 1,187 549P-Hydroxybutyrate 0 0 0 665 65 631 106Propionate 0 0 0 932 153 968 400Acetate ND ND ND 1,319 24 1,333 620Benzoate 0 0 0 713 63 902 364

a Specific dehalogenase activity is expressed as nanomoles of chloride released per minute per milligram of protein (milliunits).b ND, Not determined.

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1432 KOHLER-STAUB AND KOHLER

TABLE 4. Cofactor dependence of NADH formation with a cellextract of crotonate-grown Alcaligenes sp. strain CC1 during

incubation with aliphatic acids

nmoles ofSubstratea Cofactors added NADH/mg of

protein per min

Crotonate NAD, CoA, ATP, Mg2" 5.99-CoA 0-ATP 0-Mg2+ 0.53

3-Chlorobutyrate NAD, CoA, ATP, Mg2+ 4.09-CoA 0-ATP 0-Mg2+ 0.42

,-Hydroxybutyrate NAD, CoA, ATP, Mg2+ 44.63-CoA, ATP, Mg2+ 44.63

a With trans-3-chlorocrotonate, cis-3-chlorocrotonate, 2-chlorobutyrate, 2-chloropropionate, 3-chloropropionate, and all cofactors added, no NADHformation was observed.

dehalogenation of one chlorine, the resulting chloroethanolis first oxidized to the acid before the second chlorine isremoved.For the degradation of the P-chlorinated four-carbon fatty

acids by Alcaligenes sp. strain CC1, we propose that theactive enzyme prior to the dechlorination step, namely, anacyl-CoA synthetase, is also involved in the metabolism ofnatural compounds and exhibits activity with the chlorinatedanalogs as a result of a broad substrate specificity which isconsidered to be characteristic for the enzymes involved in,-oxidation (13). This view is supported by the observationthat dechlorination of p-chlorinated acids with resting cellsoccurs only after growth on chlorinated or unchlorinatedfour-carbon acids and that all these compounds induce anidentical or similar pathway that is not in evidence in cellsgrown on other substrates. The dechlorination reaction,which is assumed to occur on a four-carbon CoA esterderivative, is not yet understood. Most probably, the chlo-rine is removed by nucleophilic displacement with a hy-droxyl group. The enoyl-CoA hydratase, which normallyadds water to the double bond, may be involved in this

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0 50 100 ISOMINUTES

FIG. 3. Substrate consumption and chloride release during incu-bation of cell extract of crotonate-grown Alcaligenes sp. strain CCwith 1 mM trans-3-chlorocrotonate (TCC) and 1 mM cis-3-chloro-crotonate (CCC). The reaction mixture contained 1 mM TCC orCCC, 3 mg of protein per ml, 2 mM CoA, 2 mM ATP, and 1 mMMg2 . Symbols: O, TCC (mM); 0, CCC (mM); U, chloride releasedfrom TCC (mM); 0, chloride released from CCC (mM).

TABLE 5. Cofactor-dependent dechlorination of ,-chlorinatedfour-carbon acids with cell extract of crotonate grown Alcaligenes

sp. strain CC1v

Chloride SubstrateSubstrate (1 mM) Cofactors added released consumed

(mM) (mM)

trans-3-Chlorocrotonate CoA, ATP, Mg2" 0.54 0.78+ NAD 0.53 0.77- Mg2+ 0.22 0.32- ATP 0.02 0.02- CoA 0 0

cis-3-Chlorocrotonate CoA, ATP, Mg2+ 0.44 0.65- CoA 0 0

3-Chlorobutyrate CoA, ATP, Mg2+ 0.53 NDb+ NAD 0.54 ND- CoA 0 ND

a Enzyme activity was observed on the basis of disappearance of trans-3-chlorocrotonate and cis-3-chlorocrotonate and chloride release from allthree substrates (reaction time, 1 h).

b ND, Not determined.

mechanism. However, since no NAD-dependent oxidationprecedes the chloride release from 3-chlorobutyric acid, weconclude that in this case, dehalogenation occurs at asaturated carbon atom (Fig. 4). Another possibility is that amore specific dehalogenase is acting on the chlorinatedCoA-thioesters. The striking fact that Alcaligenes sp. strainCC1 already has a very specialized dehalogenase, namely,constitutive 2-haloacid dehalogenase, leads to the question

l OH

trans-3-chlorocrotonic acid

CoAATP

Cl SCoA

trans-3-chlorocrotonyl-CoA

1tH20

HCI

HO /\ SCoA

trans-3-hydroxycrotonyl-CoA

0 CSCoA

3-ketobutyryl-CoA

OH

3-chlorobutyric acidCoAATPAg+

C l SCoA

3-chlorobutyryl-CoA

1H20

HCIHO - SCoA

n

3-hydroxybutyryl-CoA

l NADH + H+

O SCoA

3-ketobutyryl-CoA

I IFIG. 4. Proposed pathways for catabolism of trans-3-chlorocro-

tonate and 3-chlorobutyrate by Alcaligenes sp. strain CCL.

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MICROBIAL DEGRADATION OF ALIPHATIC ACIDS 1433

of whether there is a correlation between the degradationpathways for a-haloalkanoic acids and the ,8-chlorinatedfour-carbon acids or whether this occurrence is a matter ofpure chance. A direct involvement of the 2-haloalkanoic aciddehalogenase in the dechlorination of 1-chlorinated CoA-thioesters does not appear to be very likely for reasons ofsubstrate specificity. Further investigations concerning theexistence and distribution among microorganisms of theability to degrade 1-chlorinated acids should provide moreinsight in the evolution and specificity of this trait.Our study on the degradation of 1-chlorinated acids has

been restricted to four-carbon acids. The only additional13-chlorinated compound tested is 3-chloropropionic acid,which proved not to be a substrate for growth, although itwas dechlorinated by crotonate-grown resting cells ofAlcali-genes sp. strain CC1 (data not shown).

Catabolism of 3-carbon acids does not necessarily involvethe 1-oxidation pathway or an initial activation by CoA.Therefore, in the case of 3-chloropropionic acid, both ap-proaches-initial dechlorination by a 2-haloacid dehaloge-nase and dechlorination after formation of a CoA-ester-might not be applicable to Alcaligenes sp. strain CC1. It isnot known yet whether long-chain 13-chlorinated aliphaticacids would be degraded by Alcaligenes sp. strain CC1,since such chemicals are not commercially available. Theabilities of this organism make it a suitable candidate forcocultural biodegradation studies with bacteria that come-tabolize chlorinated aromatic hydrocarbons, such as PCBs.

ACKNOWLEDGMENTS

This work was supported by grant ECE-8419315 from the Na-tional Science Foundation and by the University of California ToxicSubstances Program.We thank John R. Cooper for assistance with the gas chromatog-

raphy-mass spectrometry and D. D. Focht for helpful suggestions.

LITERATURE CITED

1. Bartels, I., H.-J. Knackmuss, and W. Reineke. 1984. Suicideinactivation of catechol 2,3-dioxygenase from Pseudomonasputida mt-2 by 3-halocatechols. Appl. Environ. Microbiol.47:500-505.

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