APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Nov. 1994, p. 3939-39440099-2240/94/$04.00+0Copyright C 1994, American Society for Microbiology

A Structure-Activity Study with Aryl AcylamidasesDAVID T. VILLARREAL,1* RONALD F. TURCO,2 AND ALLAN KONOPKA'

Department of Biological Sciences' and Department ofAgronomy, 2Purdue University, West Lafayette, Indiana 47907-1392

Received 6 June 1994/Accepted 16 August 1994

We examined the relationship between chemical structure and biodegradability of acylanilide herbicides byusing a set of model compounds. Four bacterial isolates (one gram-negative and three gram-positive) that grewon acetanilide were used. These soil isolates cleaved the amide bond of acetanilide via an aryl acylamidasereaction, producing aniline and the organic acid acetate. A series of acetanilide analogs with alkyl substitutionson the nitrogen atom or the aromatic ring were tested for their ability to induce aryl acylamidase activity andact as substrates for the enzyme. The substrate range, in general, was limited to those analogs not disubstitutedin the ortho position of the benzene ring or which did not contain an alkyl group on the nitrogen atom. Thesesame N-substituted compounds did not induce enzyme activity either, whereas the ortho-substituted com-pounds could in some cases.

Aryl acylamidases have been detected in plants, animals, andmicroorganisms (including fungi and bacteria) (10, 14, 16, 27,30, 44, 48). These enzymes cleave the amide bond of acylani-lide, phenylcarbamate, and phenylurea pesticides. The reactionis involved in the degradation of a wide variety of xenobiotics (6,9, 13, 23, 34).The herbicides alachlor [2-chloro-2',6'-diethyl-N-(methoxy-

methyl)acetanilide] and metolachlor [2-chloro-2'-ethyl-6'-methyl-N-(1-methyl-2-methoxyethyl)acetanilide] are, in gen-eral, not hydrolyzed by aryl acylamidases (21, 25, 29, 37). Thesetwo compounds undergo demethylations, dehydroxylations,dechlorinations, and a variety of other transformations (8).However, the fungus Chaetomium globosum formed the corre-sponding anilines from alachlor and metolachlor when exposedto these herbicides in an axenic culture (29, 43), but in eachcase the aniline was a minor fraction of the metabolitesformed.

Several researchers (5, 18, 37) have speculated that theapparent recalcitrance of alachlor and metolachlor to arylacylamidase hydrolysis may be due to steric hindrance. Thetertiary amine or the substitutions at the ortho positions on thearomatic ring might interfere with enzymatic attack on theamide bond. These researchers studied soil samples or isolatedstrains which at the most could cometabolize these herbicides.No further investigations as to which structural components ofthese compounds prevented further metabolism occurred.Their speculations have never been directly tested by a system-atic investigation of the relationship between the substitutionsfound on acetanilides such as alachlor and metolachlor and theactivity of aryl acylamidases in whole cells or cell extracts ofvarious microorganisms. Other systematic structure-activityinvestigations performed on other classes of compounds (2, 31,39) have proved beneficial in determining what molecularcomponents inhibit degradation and possibly bioremediationof contaminated sites.Here we report on such a structure-activity study involving

four acetanilide (ACT)-degrading bacteria (one gram-negativeand three gram-positive organisms) and nine substituted ACTs(Fig. 1 and Table 1). The ACTs were tested for their ability to

* Corresponding author. Mailing address: Biology Department,Trinity University, 715 Stadium Dr., San Antonio, TX 78212. Phone:(210) 736-7242. Fax: (210) 736-7229.

serve as growth substrates, and/or inducing molecules. Thephysiological characterization of these systems is valuable indirecting future attempts to isolate mutants with amidases thathave an altered substrate range.

MATERUILS AND METHODS

Isolation of microorganisms. The microorganisms used inthis study were obtained from two soils by enrichment culturetechniques. The Corynebacterium sp. strain DAK12 and theArthrobacter sp. strain MAB2 were isolated from soil at aformer pesticide disposal site at the Purdue Agronomy Re-search Center, Tippecanoe County, Ind. This site had beenexposed to various agrochemicals, including acylanilide herbi-cides. Arthrobacter strain BCL and Acinetobacter strain DV1were isolated from an agricultural soil at the Purdue Agron-omy Research Center that had been exposed to acylanilides innormal application amounts for several years. Five grams ofthe soil sample was placed into 50 ml of a xenobiotic basalmedium (XBM) (20) containing 5 mM ACT as the sole carbonand energy source. Flasks were incubated at 30°C on a shakerwater bath. Samples (1 ml each) were transferred to freshmedium when the culture appeared turbid. After severaltransfers, the cultures were streaked onto XBM plates contain-ing 5 mM ACT and onto tryptic soy agar (Difco Laboratories,Detroit, Mich.) plates. Individual colonies were purified andtested for growth with ACT as the sole carbon and energysource. The isolation of Arthrobacter strain MAB2 was previ-ously described (45) (note the taxonomic change, based onanalysis of cell wall and lipid compositions [22]).

Characterizations of strains and growth substrate range.The four isolates were characterized and taxonomically iden-tified by standard physiological and biochemical criteria (11).Growth on the various ACTs was tested by inoculating a 1:100dilution of the strains (pregrown to log phase on 5 mM ACT)into XBM broth containing the various ACTs (1 and 2.5 mM).In the cases of 2-chloroacetanilide (CIACT) and 2-chloro-2,6-dimethylacetanilide (Cl26ACT), their water solubilities did notallow these compounds to be completely dissolved for the 2.5mM concentration during these growth experiments. Growthon the herbicides alachlor and metolachlor was tested at 0.5mM only, also because of low water solubilities. The cultureswere incubated at 30°C on a rotary shaker. Cultures wereincubated up to 3 weeks before being described as having no

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Rl N C OH2 R6

R5 R2

As<R3

R4FIG. 1. Structure of model ACT compounds. See Table 1 for

specific substituents (Rl to R6) found in individual substituted ACTs.

growth; the visual appearance of turbidity was used to indicategrowth. Each strain was tested twice on each compound,except in the case of alachlor and metolachlor, which were

tested only once as growth substrates.Substrate utilization by whole cells. Substrate utilization by

washed resting cell suspensions was assayed by the ability ofACT substrates to stimulate oxygen consumption (20). Cellswere pregrown on 5 mM ACT and harvested in mid-log phase.Cells were centrifuged, washed, and then resuspended in 10mM potassium phosphate buffer (pH 7). A 1.6-ml portion ofthis suspension was incubated at 30°C, and the rate of endog-enous respiration was monitored. Most of the test substrateswere then added to a final concentration of 1 mM, and oxygen

consumption was monitored with a Clark oxygen electrode(Yellow Springs Instruments, Yellow Springs, Ohio). Theoxygen consumption rate for each substrate was linear over thetime course of the assay. The rates were corrected for endog-enous respiration. In the cases of CIACT, Cl26ACT, alachlor,and metolachlor for which the solubilities of these substratesdid not permit a final concentration of 1 mM, a 100-plIinjection of a saturated stock solution was used. Each experi-ment with each strain was replicated two to three times, and arepresentative set of data is reported here.

Substrate utilization in cell extracts. Substrate utilization bycell extracts of the four microorganisms was assayed in thefollowing manner. Cells were pregrown on 5 mM ACT andharvested in mid-log phase by centrifugation at 10,000 x g for10 min. Cells were washed in 0.1 M Tris-HCl (pH 8.6) andresuspended in the same buffer. Cells were disrupted twicewith a French pressure cell at 1,100 lb/in2. The resultingsuspension was recentrifuged at 10,000 x g for 15 min toseparate cell debris from the extract. The supernatant wasseparated and stored on ice until used. Subsamples of the cellextract were incubated with the nine test substrates (1 mMeach) in 0.1 M Tris-HCl buffer (pH 8.6). Incubations occurredat 30°C on a rotary shaker. To determine the enzyme activity in

TABLE 1. R groups of model ACT compounds

Compound Rl R2 R3 R4 R5 R6

ACT H H H H H HNMACT CH3 H H H H HNEACT CH2CH3 H H H H HClACT H H H H H Cl2ACT H CH3 H H H H3ACT H H CH3 H H H4ACT H H H CH3 H H26ACT H CH3 H H CH3 HC126ACT H CH3 H H CH3 Cl

the extract (and hence an appropriate incubation time), sub-samples were also assayed with p-nitroacetanilide as the sub-strate in a spectrophotometric assay (see below). The amountsof time needed to transform 50 and 75% of the p-nitroacet-anilide were calculated, and the flasks with the nine test ACTswere sampled at these two time points (which ranged between15 min and 3.5 h). Preliminary experiments had shown a linearrate of enzyme activity in cell extracts over the time course ofthese assays. During sampling, 1-ml portions of the reactionmixture were placed into microcentrifuge tubes and 50 ,ul of 1M HCl was added to terminate activity. Samples were frozenuntil high-performance liquid chromatography (HPLC) anal-ysis was performed. Chemical hydrolysis of the different ACTsubstrates during the incubation period was monitored in aseries of control samples which contained one of the ACTs butreceived an aliquot of buffer instead of the cell extract. Eachexperiment with each strain was replicated two to three times,and a representative set of data is reported here.

Analytical methods. HPLC was performed with a Varianmodel 5000 liquid chromatograph equipped with a GilsonHolochrome UV detector set at 210 nm. A 20-,ul portion of thefiltered assay mixture was injected into a Supelco LC-PAHcolumn by using an isocratic mobile phase consisting of 40%acetonitrile in water at a flow rate of 0.5 ml/min.Some culture solutions were analyzed by capillary gas chro-

matography with a Supelco type SPB-1 column (30 m by 0.25mm [inner diameter]) and a Varian model 3700 gas chromato-graph equipped with a flame ionization detector. The flow rateof N2 carrier gas was 4 ml/min, and rates (milliliters perminute) of gas flow to the detector were 40 for N2 (as makeupgas), 34 for H2, and 300 for air. The injector and detectortemperatures were 140 and 190°C, respectively. The columnwas operated at 70°C for 1 min, and then there was atemperature increase of 20°C/min up to a temperature of190°C. Filtered aqueous samples from microbial cultures wereextracted 1:1 with ethyl acetate, and 2-,u portions of ethylacetate extracts were injected into the column. Aqueoussolutions of aniline and ACT standards were also extracted 1:1with ethyl acetate in order to identify peaks in the experimentalsamples.A p-dimethylaminobenzaldehyde assay (7) of some samples

from the substrate range experiments was performed withcellular extracts. Product formation was determined with theauthentic anilines as standards.

Aryl acylamidase activity was assayed by monitoring theconversion of p-nitroacetanilide to p-nitroaniline, determinedby measuring A382. Reaction mixtures were incubated at 24°Cin a 1-cm cell. The reaction was monitored with a Gilfordmodel 250 spectrophotometer (Gilford Instrument Laborato-ries, Oberlin, Ohio). Reaction mixtures contained 0.5 ml of 1mM p-nitroacetanilide and 0.4 ml of 0.1 M Tris-HCI, pH 8.6.The reaction was initiated by addition of the washed cellsuspension or the cell extract. The amount of Tris-HCl bufferwas adjusted to bring the total volume to 1 ml if less than 100,u of the test material was used in the assay.

Proteins were analyzed by the method of Lowry et al. (26)with bovine serum albumin as the standard; 1 M NaOHextracts of cells or cellular material resuspended in 1 M NaOHwere heated to 100°C for 10 min and used in this procedure.

Induction of enzyme activity. Induction experiments withDAK12, BCL, and DV1 were carried out in the followingfashion. DAK12 was pregrown on 8.3 mM glycerol. BCL andDV1 were pregrown on 12.5 mM acetate. All three strainswere harvested in mid-log phase, washed, and resuspended in10 mM potassium phosphate buffer (pH 7). Cells were placedinto flasks containing 8.3 mM glycerol (DAK12) or 12.5 mM

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acetate (BCL and DV1) and 1 mM each of the nine test ACTcompounds in XBM medium. A 10th flask for each straincontained only glycerol or acetate in XB3M with no inducingsubstrate. Cells were added into each flask to obtain an opticaldensity at 600 nm of approximately 0.1. Flasks were incubatedat 30°C on a rotary shaker. After 3.5 h (DV1) or 6 h (DAK12and BCL), 1-ml samples were removed and placed intomicrocentrifuge tubes. Cells were pelleted, washed, and thenresuspended in 1 ml of 0.1 M Tris-HCl (pH 8.6). The level ofinduction by each ACT was ascertained by spectrophotometricaryl acylamidase assays with p-nitroacetanilide as the testsubstrate. Each experiment with each strain was replicated atleast twice, and a representative set of data is reported here.

Chemicals. ACT was purchased from Pfaltz and Bauer, Inc.,Waterbury, Conn. 2'-Methylacetanilide (2ACT), N-methylac-etanilide (NMACT), C126ACT,. 2',6'-dimethylaniline, and N-ethylaniline were obtained from Lancaster Synthesis, Windham,N.H. 3'-Methylacetanilide (3ACT), 4'-methylacetanilide (4ACT),CIACT, 2',6'-dimethylacetanilide (26ACT), N-ethylacetanilide(NEACT), and 4'-nitroacetanilide were all purchased fromAldrich Chemical Co., Milwaukee, Wis. p-Toluidine and m-toluidine were obtained from Sigma Chemical Co., St. Louis,Mo. o-Toluidine was purchased from the J. T. Baker ChemicalCo., Phillipsburg, N.J. N-Methylaniline was obtained fromEastman Kodak Co., Rochester, N.Y. Aniline was purchasedfrom Fisher Scientific, Fair Lawn, N.J. Alachlor and metola-chlor were obtained from Chem-Service, West Chester, Pa. Allchemicals were used as purchased.

RESULTS

Strain characterization. Strain DAK12 was a gram-positiverod and showed the typical snapping division of the genusCorynebacterium. It was catalase positive, oxidase negative, andnonmotile and grew only aerobically in fluid thioglycolatemedium. The isolate grew poorly on Simmons citrate agar,tested negative for phenylalanine deaminase, and was unableto ferment glucose. Strain BCL was a gram-positive coccoba-cillus and was catalase positive, oxidase negative, and nonmo-tile. It grew only aerobically in fluid thioglycolate medium,showed no growth on citrate, was phenylalanine deaminasenegative, and could not ferment glucose. It was thereforedesignated member of the genus Arthrobacter. Acinetobacterstrain DV1 was a gram-negative rod and was catalase positive,oxidase negative, phenylalanine deaminase negative, and non-motile. A strict aerobe, it was able to utilize citrate and toferment glucose without the production of gas.DAK12, BCL, and DV1 were initially isolated with ACT as

the sole carbon and energy source. MAB2, anArthrobacter sp.,was isolated for its ability to grow on 2-chloro-N-isopropylac-etamide (45) but was also found to be able to grow on ACT.DAK12, BCL, MAB2, and DV1 had doubling times of 3.1, 5.9,3.5, and 1.5 h, respectively, on 5 mM ACT in XBM. Gas-chromatographic analysis of ethyl acetate extracts of thestationary-phase cultures indicated the presence of aniline inthe medium and the loss of ACT. No further growth on theaniline occurred.Growth substrate range. MAB2 used more ACT substrates

as carbon and energy sources than the other strains. It grew onfive of the nine ACTs tested. MAB2 grew on ACT with nosubstitution, on ACTs with one methyl substitution on the ring(2ACT, 3ACT, and 4ACT), and on an ACT with two methylsubstitutions (26ACT). It was the only microbe able to grow onthis di-ortho-substituted compound, albeit quite slowly (dou-bling time, > 15 h). Growth on ACT and the methyl-substituted

TABLE 2. Substrate utilization by whole cells

Respiration rate (% of ACT rate) for strain:Compound

DAK12a BCLb MAB2C DVld

ACT 100 100 100 100NMACT 0 0 0 0NEACT NDe 0 0 0CIACT 19 25 16 192ACT 97 83 31 03ACT 67 93 33 04ACT 79 78 24 5326ACT 3 3 0 0Cl26ACT ND ND 0 0Acetate 68 90 91 53Aniline 0 0 0 0

a DAK12 had an endogenous respiration rate of 7.8 ,umol Of 02 per min permg of protein and a respiration rate with ACI of 102 ,umol of 02 per min per mgof protein.

b BCL had an endogenous respiration rate of 7.5 p.mol Of °2 per min per mgof protein and a respiration rate with ACT of 40 F±moI of 02 per min per mg ofprotein.

c MAB2 had an endogenous respiration rate of 6.3 F.moI Of 02 per min per mgof protein and a respiration rate with ACT of 171 ,umol of 02 per min per mg ofprotein.

d DV1 had an endogenous respiration rate of 3.2 ,umoI Of °2 per min per mgof protein and a respiration rate with ACT of 53 ,umoI Of °2 per min per mg ofprotein.

e ND, not determined.

ACTs occurred at approximately equal rates (doubling time,<5 h).The only gram-negative microbe tested, DV1, showed the

narrowest range of all four isolates. It grew on only two of thenine ACTs, ACT and 4ACT. Growth on 4ACT was relativelyslow (doubling time, >20 h). DAK12 and BCL gave identicalgrowth patterns. Both grew on four of the ACTs tested, i.e.,ACT and the three analogs with a single methyl substitution onthe ring. Both DAK12 and BCL grew on these four compoundswith doubling times of less than 10 h, with growth on ACThaving the fastest doubling time for both strains with these fouranalogs.

Notably, all four strains grew on ACT and 4ACT. None grewon the N-substituted derivatives or on the chlorinated com-pounds tested. DV1 was the only strain not able to grow on2ACT and 3ACT. None of the strains could grow on alachloror metolachlor.

Substrate utilization. Substrate utilization by whole cells wastested by the ability of substituted ACTs to stimulate oxygenuptake by ACT-grown resting cell suspensions (Table 2). Ingeneral, the range of substrates utilized was similar to thegrowth substrate range. However, chloroacetanilide was uni-formly respired by all strains, although none of them grew onit. DV1 metabolized a limited range of ACTs. MAB2 showedno ability to respire 26ACT in these experiments, even thoughit grew slowly on this compound. None of the strains couldrespire alachlor or metolachlor.The substrate range of the aryl acylamidases was tested

more directly by incubating cell extracts with 1 mM concentra-tions of the test compounds (Table 3) and measuring substratedepletion and aniline formation. The N-substituted com-pounds were not utilized as substrates by BCL, MAB2, orDV1, and activity with DAK12 was negligible. The extractsfrom DAK12 and MAB2 had low activities with the di-ortho-substituted substrate 26ACT. However, activity was completelyabolished in these extracts when the acetyl moiety was chlori-nated, as in the case of Cl26ACT.MAB2 showed about the same relative activities by both

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TABLE 3. Substrate utilization by cell extracts

Relative activity (% of ACT activity)Compound of cell extracts of straina:

DAK12 BCL MAB2 DV1

ACT 100 100 100 100NMACT 3 1 0 0NEACT 3 0 1 0ClACT 80 91 26 1192ACT 110 82 18 03ACT 132 102 10 04ACT 31 89 21 12226ACT 9 1 9 0Cl26ACT 2 0 0 0

a Specific activities with ACT (in nanomoles per minute per milligram ofprotein in the extract) for DAK12, BCL, MAB2, and DV1 are 152, 124, 25.0, and34.5, respectively.

whole cells and cell extracts on the chlorinated ACT withoutsubstitution on the aromatic ring (CIACT). The relative activ-ities were much higher in extracts than in whole-cell suspen-sions of the other three strains with CIACT. Strain DV1showed somewhat higher activity toward the ClACT and 4ACTthan toward ACT. DV1 also showed no activity toward theother methyl-substituted aromatics, 2ACT and 3ACT, thoughthe other three strains showed significant utilization of boththese compounds.

In general, the amidase of DV1 had a limited range ofsubstrates, but all of the substrates were hydrolyzed at a ratecomparable to the rate of ACT utilization. BCL and DAK12enzymes had broader substrate ranges and likewise showedgood activity against most of the substrates tested. In contrast,the MAB2 enzyme had a broad substrate range, but thesubstituted ACTs were hydrolyzed at rates substantially lowerthan those of the parent compound.

Induction of aryl acylamidase. The enzyme system for themetabolism of ACT was constitutive in isolate MAB2. How-ever, in the other three strains, aryl acylamidase synthesis wasinduced by ACT. The ability of the substituted ACTs to inducearyl acylamidase in DAK12, BCL, and DV1 was tested (Table4). In general, the substrates that induced aryl acylamidaseactivity in a strain were the same as the compounds whichserved as enzyme substrates. Several examples for which therange of inducing compounds was broader than the range ofenzyme substrates were found. Notably, 3ACT induced aryl

TABLE 4. Induction of aryl acylamidase

Relative enzyme activityCompound (nmol/min/mg of protein) of strain:

DAK12 BCL DV1

ACT 98 76 37NMACT 0 0 0NEACT 0 0 0ClACT 32 NGa 242ACT 22 74 0.83ACT 35 87 444ACT 21 82 2326ACT 23 90 0Cl26ACT 0 45 0Glycerol 0 NDb NDAcetate ND 2.1 0

a NG, no growth.b ND, not determined.

acylamidase in DV1, but it does not serve as a growth substrateor as a substrate for the enzyme. Also, Cl26ACT was agratuitous inducer for BCL. The compound 26ACT inducedrelatively high levels of the enzyme in DAK12 and BCL, eventhough this molecule was not readily cleaved.

DISCUSSION

The chloroacetanilide herbicides alachlor and metolachlorappear to be recalcitrant to mineralization by microorganisms(references 4, 21, 24, 25, 28, 32, 33, 35, 37, and 42 and thisstudy). Several reasons are possible. The most likely is that theenzymes involved in the hydrolysis of molecules similar toalachlor and metolachlor molecules may not be able to utilizethese xenobiotics as substrates. Kaufman (18) speculated thatthe N-alkyl or the ortho substituents on these herbicides mayresult in steric hindrance. Many aniline-based pesticides whichlack these specific substituents are readily degraded by micro-organisms (6, 9, 34, 41, 47).None of the organisms we tested could grow on or hydrolyze

the N-alkyl-substituted ACTs NEACT and NMACT. Thesefindings support the idea of steric hindrance by N-alkyl-substituted ACTs. Enrichment cultures with each of the nineACTs yielded no organisms which could degrade these N-substituted ACTs. In contrast, organisms which readily de-graded ACT, 2ACT, and 3ACT were easily isolated from thesesoils (19). Villarreal et al. (45) were successful in isolating anorganism that could degrade NEACT and NMACT from thesoil used here, but the organism did so by attacking the bondbetween the aromatic ring and the nitrogen atom, not byhydrolysis of the amide bond.

All four microorganisms were able to hydrolyze and respireClACT, but none were able to grow on this compound. Thismay be because the chlorinated acetate formed is toxic to themicroorganism. Alternatively, the parent compound, CJACT,might itself be inhibitory to growth as shown previously forother halogenated compounds (40). For MAB2 and DV1,further experimentation showed that (i) the cells could notgrow on chloroacetate but (ii) chloroacetanilide and notchloroacetate was toxic to these cells. Incubation of these twostrains with both 10 mM acetate and 10 mM chloroacetateshowed no inhibition of growth on acetate by the chlorinatedcompound. For both MAB2 and DV1, the presence of 2 mMClACT inhibited the growth on 10 mM acetate or 5 mM ACT.The presence of a single methyl substitution on the aromatic

ring did not limit enzyme activity, because three of the fourstrains utilized 2ACT, 3ACT, and 4ACT as enzyme substrates.But a second ortho substitution significantly reduced the abilityof these enzymes to cleave ACT. MAB2 possibly showed anability to grow on 26ACT, albeit slowly. MAB2 cultures withthis dimethylacetanilide did show some slight turbidity after 2to 3 weeks. However, this growth might be attributed tochemical hydrolysis and not biological hydrolysis of 26ACT.The activity of DAK12 extracts was significantly lower on26ACT than on 2ACT. Respiration on 26ACT by these strainswas also drastically reduced. Thus, either N-alkyl substitutionsor alkyl substitutions at both ortho positions limit the ability ofaryl acylamidase to hydrolyze the amide bond.A second possible reason for a lack of herbicide metabolism

by microorganisms may involve the inability of these moleculesto induce the appropriate enzyme systems for catabolism ofthese xenobiotics. The majority of aryl acylamidases are induc-ible systems (3, 6, 13, 15, 38, 44, 46). However, Arthrobacterstrain MAB2 had constitutive aryl acylamidase activity. TheMAB2 amidase can metabolize a wide variety of aromatic andnonaromatic acetamides (45).

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The range of compounds that induced the aryl acylamidasesin the three strains tested was very similar to the range ofcompounds that can serve as substrates. However, the induc-tion range was slightly broader in each case. This phenomenonhas also been noted in previous studies of xenobiotic metabo-lism (1, 12, 17).The di-ortho-substituted ACT 26ACT was an effective in-

ducer of aryl acylamidase of DAK12 and BCL but was not agood substrate for the enzyme. This suggests that the inabilityof these strains to grow on the di-ortho ACTs is due to theinability of the induced enzyme to hydrolyze the substrate. Incontrast, N-substituted compounds neither induce nor aresubstrates for aryl acylamidase. Thus, both factors are impor-tant for recalcitrance.A third possible reason for the lack of herbicide degradation

involves the accessibility of the substrate to the enzyme. Thetransport of xenobiotic substrates is poorly understood. Wehave no information at present on the mechanism of transport,if any, of ACT compounds into these cells.The findings outlined herein demonstrate that the recalci-

trance of alachlor and metolachlor is due to the presence of thetwo ortho substituents and the N-substituents. N-Alkyl-substi-tuted ACTs do not induce aryl acylamidases. But in soilenvironments, there might be inducing molecules present. Ifthis was the case, the enzyme would still not be able tohydrolyze these xenobiotics because the N-substituted ACTsare not substrates for the enzyme. The added factor of thedi-ortho substitutions on these molecules would contribute tothe inability to be acted upon by aryl acylamidase. An addi-tional problem for utilization of alachlor and metolachlor bymicroorganisms might be that hydrolysis of the amide bondproduces a chlorinated organic acid. Halogenated moleculesare frequently not metabolized by microorganisms.As noted previously, no microorganisms which can mineral-

ize alachlor or metolachlor have yet been isolated. If arylacylamidases such as those characterized here were to hydro-lyze these herbicides, several independent mutations wouldneed to occur. First, transcription must be induced by thepesticide itself or enzyme synthesis must be constitutive.Second, the enzyme itself must be able to allow for the N-alkylsubstitutions as well as the presence of alkyl substituents atboth ortho positions, which would possibly require two sepa-rate mutational events. The likelihood of all of these muta-tional events occurring in situ is not very high. We suggest thatthe laboratory evolution of appropriate strains able to metab-olize these herbicides would be a useful approach. A stepwiseselection of appropriate mutants in aryl acylamidase mightprove successful, as has been shown for other enzymes (1, 36).Current work in our laboratory is taking this approach. Alter-natively, novel acylanilide degradation mechanisms, such asthat described for propachlor metabolism (45), might beproductively manipulated for the degradation of compoundssuch as alachlor and metolachlor.

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