APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Apr. 1986, p. 781-7890099-2240/86/040781-09$02.00/0Copyright © 1986, American Society for Microbiology

Vol. 51, No. 4

Evolved Aniline Catabolism in Acinetobacter calcoaceticus duringContinuous Culture of River Water

R. CAMPBELL WYNDHAMDepartment ofBotany, University of Toronto, Toronto, Ontario MSS JAJ, Canada

Received 20 August 1985/Accepted 16 January 1986

Adaptation ofAcinetobacter calcoaceticus from river water to aniline depends on the dynamics of parent andmutant populations. The parent, Acinetobacter strain DON26 phenotype Anio, was common in river water andassimilated aniline effectively at micromolar concentrations, but was inhibited at higher concentrations ofaniline. The Ani° phenotype was also characterized by a broad specificity for oxidation of chloroanilines byaniline-induced cells. The mutant Ani+ phenotype was represented by DON2, isolated from a population of lessthan 100 cells ml-' in a mixed river water culture, and by DON261, isolated during continuous culture ofDON26. Ani+ strains assimilated aniline at a greater maximum specific rate than the parent and were able togrow at concentrations of aniline greater than 16 mM. These strains cooxidized phenol after growth at highaniline concentrations, but showed reduced activity toward chloroanilines. These changes plus kinetic data,oxygen uptake data, and the results of auxanography indicate that the mutant has an increased activity andaltered specificity of the initial enzyme in the aniline catabolic pathway. The parent strain, DON26, was at aselective advantage relative to the mutant at low concentrations of aniline, but was replaced by the mutant whenaniline concentrations increased. Adaptation of the mixed river water community to aniline involved selectionof both phenotypes. Reversion of the Ani+ to Anio phenotype occured at a frequency of 10-2 in the absence ofaniline selection. Plasmid content was not altered during either acquisition or loss of the Ani+ phenotype.Adaptive changes in Acinetobacter spp. populations illustrate important differences in the catabolic activities ofnatural and pollutant selected strains. They also provide a model of how natural populations may adapt topollutant stress.

An understanding of early adaptations of microbial com-munities to organic chemical exposure is critical for predict-ing rates of degradation of contaminants and for establishingguidelines for the release of organic contaminants into theenvironment. Single doses of some organic compounds inthe micromolar concentration range can adapt freshwatermicrobial populations for rapid degradation of the chemicalupon subsequent exposure (37). However, this responsedoes not occur predictably in all environments and is notwell understood at the population or molecular level. Adap-tation is also dependent on the chemical structure (36) andconcentration (8) of the contaminant. The specificity ofmetabolic activities toward chemically related organic com-pounds in mixtures also depends on prior exposure and earlyadaptation of microbial communities (17).

Studies of early adaptations are critical to our understand-ing of the technique of enrichment culture. Most often,isolates obtained by enrichment at high concentrations of anorganic contaminant are assumed to be representative ofcontaminant-degrading microorganisms in the natural envi-ronment. These environments may, however, be character-ized by very low concentrations of the contaminant and ofother essential nutrients. Under these conditions the regula-tion and activities of metabolic pathways in the indigenousmicrobiota may not reflect the metabolic capabilities ofenriched populations.The present study was initiated to determine what early

adaptations occur in the microbial communities of riverwater exposed to low concentrations of aniline. Interest inthis aromatic amine has been renewed through recent pub-lications. Lyons et al. (29) have demonstrated that the majorpathway of removal of aniline from natural waters is throughbiodegradation rather than chemical oxidation. Evidencefrom two other sources indicates that aniline degradation in

Pseudomonas spp. is plasmid encoded (1, 27). Aniline is amajor industrial chemical and intermediate which, alongwith its chlorinated and sulphonated derivatives, is used inthe manufacture of herbicides, developers, and dyes. Bio-degradation of the parent compound and the chloroanilineshas been studied in many microorganisms, including theactinomycetes (4, 39), Pseudomonas spp. (1, 20, 27, 40, 45),Paracoccus sp. (9), Alcaligenes faecalis (38), Rhodococcusspp. (2, 24), and Moraxella sp. (46).The most common pathway of catabolism is through the

action of aniline dioxygenase to form catechol and ammonia,followed by ortho-ring fission by catechol-1,2-dioxygenase(4, 20, 24, 46). There are now reports of three Pseudomonasspecies which metabolize aniline by the meta-ring fission orcatechol-2,3-dioxygenase pathway (1, 24, 27). Lyons et al.(29) established that the dominant metabolic pathway in theirpond water samples was the ortho-ring fission pathwaythrough catechol, muconic acid, and 3-ketoadipic acid.

Preliminary work on freshwater samples from a variety ofhabitats indicated that aniline-degrading strains were en-riched infrequently compared with phenol degraders. How-ever, many phenol degraders were found to adapt in thelaboratory to growth on aniline (unpublished observations;39). This work suggested that microbial adaptation to anilinein natural waters might require more complex changes inbacterial populations than simple enrichment.

MATERIALS AND METHODS

Media and strains. Minimal medium A containedK2HPO4. 3H20-KH2PO4 (10 mM), MgSO4 - 7H20 (1 mM),EDTA (0.3 mM), ZnSO4 7H20 (0.01 mM), MnSO4 .4H20(0.02 mM), CuSO4- 5H20 (0.004 mM), FeSO4 7H20 (0.1mM), and NaMoO4 .2H20 (0.004 mM) at pH 7.2. Ammonium

781

on August 6, 2018 by guest

http://aem.asm

.org/D

ownloaded from

APPL. ENVIRON. MICROBIOL.

TABLE 1. Acinetobacter strains used in this study

Strain Phenotypea Source or reference

Reference strains33305 Wild type, transformable, Ani- ATCC,b (19)33306 Trp-, transformable ATCC, (19)AH60 Ba+ Hba+ Ani- J. Trevors, University of Guelph, Canada, (22)

River water isolatesDON26 Anil Phe' NA screen of river waterDON261 Ani' Phe+ Mutant of DON26 in continuous cultureDON2 Ani+ Phe+ Aniline agar count, 225 hCDON101 Ani° Phe+ Dcar DON2 survivor of 3,4-dichloroaniline exposureDON103 Ani+ Phe- Dcar DON2 survivor of 3,4-dichloroaniline exposureDON105 Ani° Phe+ Dcar DON2 survivor of 3,4-dichloroaniline exposureDON2075 Anio Phe+ Ani° revertant of DON2a Phenotype abbreviations: Trp, tryptophan; Hex, hexadecane; Ba, benzoic acid; Hba, p-hydroxybenzoic acid; Ani, aniline; Phe, phenol; Dca,

3,4-dichloroaniline.bATCC, American Type Culture Collection.c River water continuous culture sampling time.

sulfate and carbon sources (0.2-p.m filter sterilized) wereadded to give 5 mM nitrogen and 50 mM carbon unlessotherwise indicated. Aniline was used as a source of bothcarbon and nitrogen.

Inocula for batch and continuous culture experimentsconsisted of overnight cultures in 5 ml of L broth (16)incubated with shaking at 22°C.Acinetobacter calcoaceticus reference strains are listed in

Table 1 along with strains isolated in this study. The latterwere subjected to standard identification test procedures(16). Strains were plated on EMB (Levine) agar (32) andwere tested for growth with various carbon sources at 50mM C on medium A, which contained 1.8% agar. Tests forgrowth on selected aromatic carbon sources were carried outby auxanography (33) also on medium A. Strains were

compared by using rapid identification biochemical andantibiotic test panels (Sceptor; Becton Dickinson and Co.) togroup isolates into like strains. All tests and incubationswere carried out at 22°C. DNA extracts of selected riverwater isolates and reference strains were tested for theirability to transform A. calcoaceticus ATCC 33306 (trpA23)to prototrophy (22).

Screening of culturable bacteria for aniline metabolism.During the continuous culture of river water, spread platesfor the determination of culturable bacterial counts on nu-trient agar (NA) were used as a source of isolates. Stockcultures were obtained by purification on NA of 140 of themost abundant colony types from dilutions of each of theriver water and continuous culture samples. A heavyinoculum of all isolates was tested on aniline (8.3 mM) platesfor growth. Growth and the appearance of visible diffusionproducts were assessed after 3, 7, and 14 days. Selectedstrains were tested for growth at low concentrations ofaniline by auxanography, as described above. Strains main-tained for detailed work are listed in Table 1 along withisolates and mutants selected on aniline initially. Anilinephenotypes are designated Ani+ for growth on aniline-agarat 8.3 mM aniline and Ani0 for growth on aniline in contin-uous culture at concentrations in the micromolar range andinhibition of growth at millimolar concentrations of aniline.

Continuous culture. Samples of 20 liters of water contain-ing disturbed material from the sediment-water interfacewere taken in sterile bottles from the Don River at theGlendon campus of the University of Toronto. The contin-uous culture apparatus consisted of an LH fermentor with aworking volume of 1.2 liters, agitator, and oxygen and

temperature controls. The inoculum was 800 ml of the riverwater with suspended sediment. A subsample was dilutedfor plate counts on NA and aniline agar. The river water wasalso analyzed for ammonia and aniline background concen-trations and for turbidity at a wavelength of 600 nm (A600).Ammonia was determined by the formation of indophenolblue (A630) (41). Aniline was determined by diazotization andcoupling to N-(1-naphthyl)ethylenediamine dihydrochloride(10, 35).The remaining sample was centrifuged (6,000 x g, 30 min)

to remove particulates and then autoclaved (121°C, 45 min)in 15-liter volumes. Continuous dilution began within 8 h ofsampling, at a rate of 0.1 h-1. After stabilization for 2 days,20 ,uM aniline was added to the sterile reservoir and theculture vessel. Samples were removed at 9:00 a.m., 1:00p.m., and 5:00 p.m. daily for determination of A600 andconcentration of NH3 and aniline and for plate counts on NAand aniline agar.Continuous culture of the isolate DON26 was carried out

in half-strength medium A plus NH3 (0.17 mM), yeast extract(0.001%, wt/vol) and acetate (2.5 mM). This medium is Nlimited with a C/N ratio of about 20:1. Acinetobacter strainDON26 was cultured for 24 h at a dilution rate of 0.05 h-1,and then 0.1 mM aniline was added to the reservoir andculture. The culture A600 and NH3 and aniline concentrationswere determined at intervals as described above. Catecholwas determined by the method of Arnow (3). The metabolitewas identified as catechol by extraction (5) and thin-layerchromatography on silica gel G plates in a solvent system ofchloroform-10% methanol-1% acetic acid with an authenticstandard. Samples of the culture were also removed fordilution plate counts on NA and aniline agar. This culturewas continued for 265 h (13 generations). The reservoirconcentration of aniline was then raised to 5 mM, and thedilution rate was increased to 0.09 h-1. DON26 was culturedunder these conditions for 175 h (16 generations). For thefinal 185 h (17 generations) of the DON26 culture, the anilineconcentration in the reservoir was returned to 0.1 mM, andfree NH3 was eliminated from the medium.

Kinetics of aniline assimilation. Samples taken at intervalsfrom the continuous culture of isolate DON26 were washed(6,000 x g, 20 min), suspended in medium A to a density of106 viable cells ml-' as determined by NA plate count, andaerated for 1 h to eliminate aniline. [U-14C]-aniline (ICNPharmaceuticals Inc.; 10 mCi mmol-1) in filtered 0.17 mMHCI was added to 3-ml diluted subsamples of the culture in

782 WYNDHAM

on August 6, 2018 by guest

http://aem.asm

.org/D

ownloaded from

EVOLVED ANILINE CATABOLISM 783

serum-stoppered 15- by 125-mm test tubes at concentrationsfrom 0.1 to 30 puM. Acid (20 mM HCI)- and HgCl2 (10mM)-killed controls were preincubated for 30 min before theaddition of the substrate. After a further 20 min, incubatedsamples and controls were filtered (0.2-p.m pore size, 25-mmdiameter; Nuclepore Corp.) to determine the amount ofsubstrate assimilated into the cells as previously described(43, 44).Two methods of determining kinetic constants were used.

The assimilation data were analyzed, assuming Michaelis-Menten saturation kinetics, by using Langmuir-Hanes plotsof slv against s (where s is the initial aniline concentration inmicromoles per liter and v is the rate of assimilation inmicromoles per liter per hour). The data were analyzed bylinear regression to determine KA, the half-saturation con-stant, and VA, the maximum assimilation rate, with confi-dence intervals. VA was then expressed as the maximumspecific activity based on the viable cell count. The secondmethod assumed assimilation was proportional to both s andthe cell concentration (XA) in a second-order reaction (11).The specific affinity aA (liters per cell per hour) was deter-mined from the initial slope of a plot of specific rate (v x

XA-', micromoles per cell per hour) against s (micromolesper liter).

Resting cell experiments. Strain DON2 was grown on Lbroth or medium A plus aniline and washed once withmedium A. The cells were suspended in medium A to adensity of ca. 108 cells ml-'. Dilution counts were done onNA, aniline agar, and succinate-NH3 agar. The cells wereshaken, and 0.25 mM 3,4-dichloroaniline (Aldrich ChemicalCo., recrystallized) was added to each culture at 1 h. Viablecounts were determined at 3-h intervals for 25 h. At the endof the experiment, surviving clones on both NA and anilineagar plates were tested for aniline phenotype and growth onphenol.Uptake of oxygen by resting cells was determined with an

Orion polarographic oxygen electrode. Cells were centri-fuged twice from medium A, suspended to an A6wo of 0.5, andaerated until the endogenous uptake was constant. Theendogenous rate was subtracted from measured rates ofoxygen uptake with various substrates at 2 mM concentra-tion. Cell protein concentrations were determined by themethod of Lowry et al. (28).Whole cell activities and activities in cell-free extracts of

catechol oxygenases were determined spectrophoto-metrically (7) by using the extinction coefficients for theproduct of catechol-2,3-oxygenase (2-hydroxymuconic acidsemialdehyde) of Bayly et al. (6) and for the product ofcatechol-1,2-oxygenase (muconic acid) of Hegeman (19).Activities are expressed on the basis of protein concentra-tions (28). Cell-free extracts were prepared by lysis of a cellpaste with 0.1 mg of lysozyme (Sigma Chemical Co.) per mlin 10 mM EDTA-1 mM dithiothreitol-50 mM Tris buffer (pH8.0) followed by nuclease treatment. Cell debris was re-moved by centrifugation at 27,000 x g for 40 min at 4°C. Fordeterminations of catechol-1,2-dioxygenase activity, somecell-free extracts were dialyzed against 25 mM Tris (pH 7.5)containing 1 mM mercaptoethanol-0.5 mM FeSO4, followedby dialysis against the same buffer containing 0.05 mMFeSO4.

Plasmid and protein electrophoresis. Polyacrylamide gelelectrophoresis was carried out with cell-free extracts ondiscontinuous, nondenaturing, 7 to 15% gradient-densitygels (13). Plasmid isolations were carried out on overnight Lbroth cultures by a rapid sodium dodecyl sulfate lysismethod (30) modified by substituting Sarkosyl for sodium

A600.1

logCFU(mi)

0[Aniline](uM)

FIG. 1. Continuous culture of river water. A, Culture density (0)and aniline concentrations (0). Aniline was added to the reservoirand the culture at 54 h. B, Plate counts on samples taken at the timesindicated by the arrows. Total counts on NA (left bar) and anilineagar (right bar) are indicated at each sample time.

dodecyl sulfate and by eliminating the 5 M NaCl precipita-tion step. Preparations were purified by density gradientcentrifugation in CsCl (30). In addition, three methodsdesigned for isolation of high-molecular-weight plasmidswere used as described previously (18, 23; in-well lysismethod in reference 34). Electrophoresis of DNA was car-ried out in 0.75% agarose-40 mM Tris-acetate-2 mM EDTA(pH 7.9) at 1.5 V cm-' overnight and stained with 1 pug ofethidium bromide per ml. Determination of covalentlyclosed circular, open circular, and linear forms of plasmidswas by two-dimensional agarose gel electrophoresis incor-porating a UV nicking step (21). HindIII-digested (30) Xphage DNA and covalently closed circular plasmids ofAcinetobacter strain AH60 (25) were used as molecularweight markers.

RESULTS

Continuous culture of river water. Aniline was removed ata rate of 0.34 p.mol liter-' h-' from river water after a lagperiod of 24 h (Fig. 1A). Thereafter, aniline in the sterileriver water feed was removed to below the detection level of2 p.M in the culture vessel. There was no significant loss ofaniline from the reservoir during the same interval. Theinitial decline in culture density from A6N 0.03 to 0.01 in thefirst 50 h of continuous culture was due to the washing out ofparticulate matter in the inoculum. Thereafter nutrients inthe river water and nutrients released by sterilization of thewater supported a mixed population in the culture vessel of5 x 106 bacteria ml-1, about the same density as in the riverwater. Free NH3, at 30 p.M in the sterile river water, wasassimilated, leaving undetectable amounts (<5 p.M) in theculture.The results of plate counts on aniline agar of samples of

the inoculum and the continuous culture are given in Fig. 1B.The inoculum (1-h) sample and the 72-h sample containedfew bacteria capable offorming colonies on aniline agar. Theplates were incubated for 11 days before the counts shown

VOL. 51, 1986

on August 6, 2018 by guest

http://aem.asm

.org/D

ownloaded from

APPL. ENVIRON. MICROBIOL.

TABLE 2. Michaelis-Menten kinetic constants for anilineassimilation

KAbqMO Spact'(10-v SpecificStrain Culture Kiter-) ,umolcell-t affinity (aA)cStan samplea liter-') ~mlcl' (10-1 liters

h-1) cell-' h-1)

DON26 265 h 0.26 + 0.30 0.20 ± 0.05 0.52 ± 0.14DON261 444 h 0.88 ± 0.47 0.98 ± 0.36 0.78 ± 0.29DON261 636 h 0.43 ± 0.37 0.53 ± 0.09 0.34 ± 0.06DON2 Batch 0.31 ± 0.22 0.05 ± 0.02 0.06 ± 0.02

a Times indicate the hour of sampling of continuous culture in Fig. 2.b Variations are 95% confidence intervals.c Variations are standard errors.

(Fig. 1B) of 102 bacteria ml-' (two colonies on 20-,u spreadplates of undiluted water) were recorded.During and after aniline adaptation (121 h, 225 h), the

aniline CFU increased to 104 bacteria ml-'; these coloniescould be counted after 2 days. All aniline agar colonies weresimilar: cream-brown, smooth, circular, and with a diffus-able black color. In all cases, enriched aniline-degradingisolates gave test results as summarized for the representa-tive isolate DON2 and were indistinguishable in reactions ofthe Sceptor biochemical-antibiotic test panels. Test resultsfor strains DON26 and DON2 were as follows: gram nega-tive (some cells resisted decolorization); coccobacillus;nonmotile; cream-brown colonies on NA; blue colonies onEMB agar; oxidase negative; catalase positive; no growth inanaerobic conditions; growth at pH 5.6; growth on suc-cinate, acetate, hexadecane, octane, 2,3-butanediol, phenol,and benzoic acid; formation of mutant colonies on p-hydroxybenzoic acid during auxanography; no growth onglucose or citrate; transformation of ATCC 33306 (comple-mentation of trpA23 mutation by DNA from DON isolates[19]). At a frequency of 0.4% of culturable bacteria at 225 h,oxidase-negative colony types identical to DON2 were theonly colony types formed on aniline agar.

Screening of river water isolates on aniline agar. Isolatesselected from NA plate counts at the times indicated in Fig.1B were purified on NA and screened for growth on anilineagar. Of 140 isolates from river water, none was capable ofgrowth on aniline within the usual 3-day incubation period.However, three isolates gave rise to mutant Ani+ colonieson aniline agar within 5 to 11 days. The Ani+ mutantfrequency of these isolates in controlled dilution experi-ments was 2.5 x 10-4.The isolates exhibiting Ani+ mutant phenotypes were

identical based on the taxonomic criteria listed above for therepresentative isolate DON26. They were also indistinguish-able in all but aniline phenotype from the isolates purifiedfrom aniline agar throughout the continuous culture experi-ment. The polyacrylamide gel electrophoresis protein band-ing patterns for DON26 and DON2 grown on succinate inbatch culture were identical. The taxonomic criteria identifythese strains as A. calcoaceticus, (phenon 1; 32). To confirmthis identification, DNA prepared from strains DON2 andDON26 was used to transform a tryptophan auxotroph of A.calcoaceticus (ATCC 33306) by the method of Juni (22).Transformation in Acinetobacter spp. is an effective crite-rion for identification. The frequency of transformation byDNA of the DON strains was 1 order of magnitude lowerthan for the DNA of the isogenic prototroph A. calcoace-ticus (ATCC 33305); however the results were reproducibleand confirm the identification based on taxonomic criteria.The frequency of isolates exhibiting the Ani+ mutation

within the total, NA-culturable community increased from2% in the river water to 12 and 32% after 121 and 225 h ofcontinuous culture, respectively.On the basis of the results obtained with the mixed river

water culture, another continuous culture experiment withDON26 in isolation was initiated. The experiment wasdesigned to show that Acinetobacter strain DON26 wascapable of mutation to an Ani+ phenotype in continuousculture under conditions of limiting aniline N. Contrary toexpectation, the parent strain DON26 was able to removeaniline from the medium to support growth when the anilineconcentration was very low (less than the detection limit of2 puM in the culture vessel) (Fig. 2). The number of Ani+mutants increased to a frequency within the parent popula-tion of 1.6 x 10-2 between 25 and 31 h when aniline wasbeing removed from the culture. After that the frequencydeclined to approximately io-4 (Fig. 2B). This is comparableto the frequency in Acinetobacter strain DON26 in theabsence of selection. This frequency of Ani+ mutants in theparent population was maintained for 250 h with no tendencyfor the mutant to displace the parent population.At 300 h the reservoir concentration of aniline was raised

to 5 mM, resulting in an increase of the aniline concentrationin the culture to almost 1 mM (Fig. 2A). Culture densitydropped during the next 24 h. A toxic effect of the anilinewas reflected in the number of parent and mutant strainsenumerated by plate count at 300 h (Fig. 2B). Rapid growthof the mutant was observed in the next 48 h, resulting in aculture density that fluctuated between A6w 0.3 and 0.5 andelimination of the parent strain from the culture. With theincrease in culture density, aniline concentrations decreasedto less than 2 R,M, and a transient accumulation of catecholto almost 1 mM occurred (424 h, Fig. 2A). When the culturedensity reached its maximum at 472 h, there was no detect-

.6

.5

0

A600.3

.2

.1

8

lo9 64CFU(mri)4

2

100 200 300 400Time (h)

600

10000

(Aniline](M)

600a

[Catechol]

200

FIG. 2. Continuous culture of A. calcoaceticus DON26. A,Culture density (0), aniline (0), and catechol (O) concentrations inthe culture. The reservoir concentration of aniline was increasedfrom 0.1 to 5.0 mM at the time indicated by the first arrow and thenreduced to 0.1 mM with elimination of NH3 in the reservoir at thetime indicated by the second arrow. B, Plate counts on NA (0) andaniline agar (0) during culture.

A.

100 200 300 400 500 - 600Time (h)

71

784 WYNDHAM

on August 6, 2018 by guest

http://aem.asm

.org/D

ownloaded from

EVOLVED ANILINE CATABOLISM 785

TABLE 3. Oxygen uptake rates and rates relative to aniline uptake for resting-cell suspensions

Oxygen uptake rate, ,umol min-' mg-' (%), on substrateAcinetobacter

strain Acetate Aniline Catechol Phenol 3,4- 4Dichloroaniline Chloroaniline

DON26a 1.98 (129) 1.53 (100) 2.30 (150) 0.00 (0) 0.62 (41) 0.30 (20)DON261a 1.86 (53) 3.49 (100) 2.91 (83) 0.00 (0) 0.10 (3) 0.07 (2)DON261b 1.58 (22) 7.23 (100) 4.29 (59) 1.02 (14) 0.16 (2) 0.13 (2)DON2C 2.33 (50) 4.68 (100) 4.80 (103) 0.83 (18) 0.24 (5) 0.19 (4)

a Cells grown in continuous culture with aniline as limiting source of nitrogen at 0.1 mM in the reservoir. Dilution rate, 0.09 h-1.b Cells grown in continuous culture with aniline as limiting source of carbon at 5 mM in the reservoir. Dilution rate, 0.09 h-'.c Cells grown in batch culture on 25 mM acetate plus 2 mM aniline.

able. aniline or catechol present in the culture. At 474 h thereservoir concentration of aniline was reduced to the previ-ous level of 0.1 mM, and free NH3 in the medium waseliminated. The culture density immediately dropped to anA6w of 0.05 and was stably maintained for a further 150 h.There was no replacement of the mutant by the parent strainduring this time. At 600 h the Ani+ mutant of DON26 wasisolated and designated DON261.

Kinetics of aniline assimilation. At three times during thecontinuous culture of Fig. 2, when strain DON26 (265 h, lowaniline), DON261 (444 h, high aniline), or DON261 (636 h,low aniline) dominated, samples were removed and dilutedto 106 bacteria ml-1 for the determination of aniline assimi-lation kinetics. The results for kinetic constants determinedon the basis of a Michaelis-Menten treatment of the data andspecific affinities based on a second-order treatment aregiven in Table 2. The results are also shown for DON2 grownin batch culture with aniline as a carbon and nitrogen source.There was no significant difference in the half saturationconstants or the specific affinities for the parent and mutantstrains growing in continuous culture. The maximum specificactivity for aniline assimilation by D0N261 grown at highaniline concentration was fivefold higher than for DON26grown under identical conditions at limiting aniline concen-trations. The difference was 2.5-fold when comparingDON261 grown at low concentrations of aniline. The resultsfor DON2 grown in batch culture on 8.3 mM aniline show alow maximum activity and specific affinity. The cells usedfor this sample were in the late exponential phase of growthand probably included a large number of cells with repressedactivity.On the basis of the kinetic results, an Anio phenotype was

assigned to the river water isolate DON26, signifying that theparent strain, while unable to compete with the Ani+ mu-tants at high concentrations of aniline or form colonies on 8.3mM aniline agar, still effectively assimilated aniline atmicromolar concentrations.

Resting cell experiments. The nature of the mutation ofDON26 to DON261 was investigated further by determiningoxygen uptake rates for resting cells from continuous andbatch cultures. The aniline oxygenase system was inducible,with no activity in acetate-grown cells. Growth in continu-ous culture with aniline as the limiting source of nitrogeninduced a nonspecific oxygenase activity in DON26, whichoxidized chloroanilines (Table 3). Only slight activity towardchloroanilines was observed in the mutants grown under Nlimitation or at high aniline concentrations. For growth inliquid culture on succinate plus NH3, the MIC of 3,4-dichloroaniline was 0.06 mM for DON26 and 0.3 mM forDON261. The greater toxicity of dichloroaniline towardDON26 may be because it is metabolized at a greater rate inthe parent. There was no uptake of oxygen when DON26

and DON261 grown at low aniline concentrations wereexposed to phenol (Table 3). When the aniline concentrationwas raised to 5 mM in the DON261 reservoir, or when DON2was grown in batch culture on 2.0 mM aniline, oxygenuptake on exposure to phenol was observed.The continuous culture experiment with strain DON26

(Fig. 2) indicated that once selection of the mutant had beenachieved by elevating the aniline concentration, a return to alow aniline concentration did not select the parent strainagain. Although this experiment provided no evidence forreversion, results from other work show that reversion of theAni+ to Anil phenotype can occur at high frequencies. Afterthree transfers of DON2 under nonselective conditions in Lbroth and plating on NA, in independent experiments, 2 of46 and 2 of 152 clones exhibited spontaneous loss of the Ani+phenotype. When these revertants (designated DON2075)were then tested for their ability to again acquire the Ani+phenotype, frequencies of acquisition were similar to that ofthe original river isolate DON26.

Revertants were also observed after exposure of Ani+strains to toxic levels of chlorinated anilines. Figure 3illustrates the effect of 0.25 mM 3,4-dichloroaniline on theviable counts of resting DON2 cells grown under noninduc-ing and inducing conditions. When cells were grown on Lbroth, 3,4-dichloroaniline had no effect on either the totalcount (on NA) or the aniline agar count throughout the 25-hexposure. When cells were grown on aniline, exposure to0.25 mM 3,4-dichloroaniline did not effect the total count (onNA) for the first 16 h; however, during that period cells ableto form colonies on aniline agar decreased by 3 orders ofmagnitude. Colonies developing on NA plates between 10and 16 h from the aniline-induced suspension were tested forgrowth on aniline agar. All colonies retained the Ani+phenotype, indicating that the decline in viable counts onaniline agar was due to short-term toxicity of aniline or ametabolite. This toxicity was not observed in the L broth-grown cells and could be reversed in aniline-grown cells bysubculturing on NA. A possible explanation is thatdichloroaniline or a metabolite inactivates one of the en-zymes in the catabolic pathway, causing accumulation oftoxic levels of an aniline metabolite when these cells aretransferred directly onto aniline agar.More interestingly, the surviving fraction of the aniline-

induced resting cell population isolated after 24 h (Fig. 3)was highly variable in phenotype. The survivors fell intothree categories, represented by strains DON101, DON103,and DON105 (Table 1). DON101 was indistinguishable fromthe parent strain DON26. DON103 formed large amounts ofcatechol during growth on aniline and failed to grow onphenol as a carbon source at any concentration, as deter-mined by auxanography (Table 4). DON105 had an AnioPhe+ phenotype, similar to the parent strain DON26, except

VOL. 51, 1986

on August 6, 2018 by guest

http://aem.asm

.org/D

ownloaded from

APPL. ENVIRON. MICROBIOL.

Time (h)

FIG. 3. Counts of surviving DON2 resting cells on exposure to0.25 mM 3,4-dichloroaniline. L broth-grown cells were washed andsuspended to about 5 x 10' bacteria ml-t, 3,4-dichloroaniline wasadded at 1 h, and samples were taken for total counts on NA (O) oraniline agar (U). Aniline-grown cells were similarly resuspended toabout 108 bacteria ml-' and exposed to 3,4-dichloroaniline, andsamples were taken for NA (0) and aniline agar (0) counts.

that DON105 was more sensitive to both phenol and anilineinhibition (Table 4) and did not acquire the Ani+ phenotypeat a detectable frequency. The high frequency of isolation ofcatabolic mutants on exposure of aromatic compound-degrading strains to chlorinated substrate analogs has beendescribed previously (42).The highly variable phenotypes observed in the Acineto-

bacter strains prompted tests on the activity of catechol-1,2-dioxygenase. This enzyme could be detected in whole cells;however in cell-free extracts little or no activity was de-tected. Other catechol oxygenases have been shown to beiron-sulfur proteins in which the active iron may be labile(26). When cell-free extracts of DON26, DON261, andDON2 were dialyzed against FeSO4 under reducing condi-tions overnight, the activity of catechol-1,2-dioxygenase wasrestored to 20 to 50 nmol min-1 mg-1. There was noconsistent difference between the dialyzed catechol-1,2-dioxygenase activities of the three strains. There was also nodifference in the sensitivity of these strains and DON103 andDON105 toward catechol in diffusion plate tests. The diam-eter of growth inhibition zones in auxanography (Table 4)show that all strains exhibit the same response to catechol.

Significant differences occur in the utilization of anilineand phenol by these strains. Growth inhibition zones for theparent DON26 strain confirm the results found in continuousculture, that aniline is toxic at high concentrations. Phenol isless toxic at an equivalent initial concentration on the plate.The Ani+ strains DON261 and DON2 show a reversal of therelative toxicities of aniline and phenol when compared withthe parent strain. Two of the surviving strains isolated afterexposure to 3,4-dichloroaniline, DON103 and DON105, alsohave altered aniline and phenol growth phenotypes as de-scribed above.Plasmid screening of Acinetobacter isolates. The high fre-

quency of spontaneous loss of the Ani+ phenotype in DON2prompted a plasmid screen. DON2 contains three plasmidsof molecular size 3.5, 5.3, and 6.8 megadaltons as deter-mined by electrophoresis with marker plasmids of A. calco-aceticus AH60 (25) (Fig. 4). The positions of open circularforms of these plasmids are indicated. A restriction enzymedigest of a total plasmid preparation of DON2 indicated the

small plasmid contains one HindIII site, whereas the largerplasmids contain multiple sites. No high-molecular-weightplasmids have been detected in this strain by alkaline dena-turation (18) or in-well lysis methods (34). The plasmidprofiles of the Anio parent and a spontaneous Ani+-to-Ani°revertant (DON2075) are also given in Fig. 4. There is nosignificant difference in the covalently closed circular andopen circular plasmid banding patterns and apparent copynumbers for these strains. Hindlll restriction digest patternsfor the two phenotypes were identical to that shown forDON2 (Ani+).

DISCUSSIONAt least 2% of the culturable heterotrophic community of

bacteria in unamended Don River water carried the aromaticpathway genes required for the utilization of Rtniline. Withinthe limitatipns of the screening procedure, only Acineto-bacter strains were found to assimilate aniline as a carbon,nitrogen, and energy source in these samples. Other popu-lations in the mixed river water community may haveassimilated aniline at low concentrations but may have beennonculturable. Although Acinetobacter spp. have not beenreported in the past to assimilate aniline, parallel aromaticcatabolic pathways for the assimilation of benzoate andp-hydroxybenzoate have been well characterized (12). Mem-bers of the related genus Moraxella assimilate both anilineand chloroanilines (46).There were two modes of expression of the aniline growth

phenotype which occurred in unamended river water. Thedominant phenotype under these conditions was Ani°, ex-pressed by the representative strain DON26, which wascharacterized by a low half-saturation constant and highspecific affinity for aniline. The assimilation system in thisstrain was saturated at concentrations of aniline of 2.6 ,uM(10 times the KA). Growth was inhibited at millimolarconcentrations of aniline. The Ani° phenotype was presentin numbers of 3.6 x 104 ml-' of river water.A much smaller population in the river water, estimated at

less than 100 cells ml-', exhibited the Ani+ phenotype. Thisphenotype was readily detected by the ability of cells togrow on concentrations of aniline up to 16 mM on solidmedia.

Identification test procedures, polyacrylamide gel electro-phoresis protein banding patterns, and plasmid profiles dem-onstrated that isolates representative of the Ani° and Ani+Acinetobacter spp. phenotypes were identical in all respectsexcept their ability to grow on aniline and phenol. Theseresults suggested that within the Acinetobacter population of

TABLE 4. Growth inhibition zones after auxanography withAcinetobacter strainsa

Growth inhibition zone diameter (cm) on carbon source:Strain

Aniline Phenol Cate- Benzoate p-Hydroxy-chol benzoate

DON26 3.2 1.0 1.8 0.3 _bDON261 0.5 2.5 1.9 0.2 -DON2 0.5 2.3 2.0 0.2 -/+cDON103 1.0 - 2.0 0.2 -DON105 >4 3.2 1.9 0.3 -

a Each substrate (30 ,umol) added to the center of a spread plate of eachstrain of approximately 107 bacteria. Growth inhibition zone diameter wasdetermined after 3 days of incubation at 25°C.

b No growth.c Mutant of DON2 isolated which grows well on p-hydroxybenzoate.

786 WYNDHAM

on August 6, 2018 by guest

http://aem.asm

.org/D

ownloaded from

EVOLVED ANILINE CATABOLISM 787

the river water, a mutant Ani+ phenotype occurred at afrequency of about lo-4. This phenotype provided a selec-tive advantage to the mutant in relation to the parehit and tothe heterotrophic community as a whole when aniline wasadded to the river water.

In continuous culture experiments it is difficult to deter-mine whether a single mutation is responsible for populationshifts, since periodic selection is continually tuthing overresident strains (14). The problent is made more complexwhen a number of competing populations are present in amixed cotnmunity. In isolation, Acinetobacter strain DON26gave rise to mutant colonies on aniline agar at a frequency oflo-, which is within the range of frequencies expected forforward point mutations or rearrangements. However, untilthe genetic basis of these variations is known and a specificmutation can be demonstrated to occur in the mixed com-munity, the Ani° and Ani+ populations in river water must beconsidered to vary independently.There was no tendency for the Ani+ phenotype to replace

the Anio population during 225 h of continuous anilineaddition to the river water. The frequency of DON2 relativeto DON26 was main'taihed at about 10-2. This is greater thanthe nonselected frequency of DON261 in the DON26 parentpopulation ahd the frequency in continuous culture onlimiting concentrations of aniline, both about lo-4. Thereason for this is unclear. A possible explanation is thatcommunity interactions in the mixed river water culturefavor a higher frequency of the Ani+ phenotype. For exam-ple, different toxic responses to added aniline may influencethe competitive advantage of the Anio and Ani+ phenotypesrelative to the heterotrophic community as a whole. t:ON26is at least 30 times more sensitive to the toxic effects ofaniline than is the mutant.The results of the continuous culture of DON26 demon-

strated the influence that kinetic properties of cells forassimilation of organic substrates have on the outcome ofcompetition experiments. At concentrations of aniline lessthan 2 p.M in the chemostat, the parent Anio strain was at aselective advantage. Half-saturation constants and specificaffinities for the substrate were not significantly different forthe Ani° and Ani+ strains. However, due to the difficulties ofmeasuring very low atmounts of the substrate and of deter-minirng the number of actively assimilating cells, there wasconsiderable variation in the determined constants. tDiffer-ences in growth rate of as little as 0.005 generation h-1 as aresult of small differences in kinetic constants can determinethe outcome of competition between mutants (14).At concentrations of aniline greater than the half-

saturation constant (0.2 to 0.9 ,uM), the maximum rate ofaniline assimilation will influence the coMpetitive outcome.DON261, with a maximum specific activity 5 times that ofthe parent, displaced DON26 when this condition was im-posed in the continuous culture.

Spain et al. (37) observed an increase in numbers ofp-nitrophenol degraders of 2 orders of magnitude, corre-sponding to a similar increase in rates of degradation of thesubstrate in experimental ponds exposed to 2 ,uM p-nitrophenol. They observed extended lag periods of up to 19days before rapid proliferation of the p-nitrophenol-degrading population. They did not suggest a controllingfactor in the length of the lag phase, but they suggested thatit was unlikely that mutation or recruitment of plasmid geneswas involved because lag periods, population changes, andspecific bacteria present after adaptation were similar amongtheir various test systems. The results presented here indi-cate that mutation may be involved in the adaptation of

FIG. 4. Plasmid electrophoresis. Lanes: 1, A. CalcoaceticusAH60 (25) with covalently closed circular plasmid markers of 140,7.9, 5.0, 4.3, 3.3, 2.7, and 2.5 megadaltons; 2, DON2 (Ani+) withcovalently closed circular (-) and open circular (>) plasmids of 6.8,5.3, and 3.5 megadaltons; 3, HindII digest of the three DON2plasmids with linear fragment sizes of 3.5, 3.3, 2.2, 1.9, 1.6, and 0.7megadaltons; 4, Hindlll digest of k DNA; 5, DON26 (Anio); 6,DON2075 (Ani° revertant).

microorganisms to even simple aromatic compounds. In theDon River waters the putative parent and mutant popula-tions were both involved in the assimilation of aniline andwere both selected. Population dynamics of a parent andmutant with very different activities for the assimilation of anorganic compound may have a correspondingly greaterinfluence on the control of lag periods and ultimate rates ofdegradation.The Acinetobacter strain DON26 mutants DON261 and

DON2 metabolize aniline to catechol and cis,cis-muconate.It is reasonable to assume from the oxygen uptake data thatthe parent strain uses the same catabolic pathway. Theobserved isolates from the river water culture and DON26mutants isolated in pure culture exhibited several propertieswhich suggest that the mutation affects the first step in thecatabolism of aniline. Kinetic data for aniline assimilationindicated that DON261 had a fivefold higher maximumspecific activity for aniline assimilation compared withDON26. The oxygen uptake data confirmed this observa-tion, with rates twofold higher for cells grown at lowconcentrations of aniline and fivefold higher in the mutantgrown at high concentrations of aniline. The oxygen uptakeexperiments showed that chlorinated anilines were oxidizedat substantially greater relative rates in the parent than in themutants, indicating a change in specificity of the oxygenase.This change was also evident in the cooxidation of phenol byaniline-induced mutant strains grown at high aniline concen-trations. The results of auxanography pointed to changes inthe aniline and phenol growth phenotypes of the mutants,whereas growth on catechol and benzoate was unaffected.Catechol-1,2-dioxygenase activity appeared not to be alteredsignificantly in the induced strains. All of these observationssuggest that the activity which is changed during adaptationof Acinetobacter strain DON26 to aniline is that of anilinedioxygenase.

Spontaneous reversion of the Ani+ to Anio phenotypeoccurred at a high enough frequency (10-2) to suggest theinvolvement of plasmid-encoded activities. Cryptic plasmidsare present in the DON isolates; however there was nocorrelation between the acquisition and loss of the Ani+phenotype and the plasmid complement. These plasmids aresmall compared with other known catabolic plasmids. Cryp-

VOL. 51, 1986

on August 6, 2018 by guest

http://aem.asm

.org/D

ownloaded from

APPL. ENVIRON. MICROBIOL.

tic plasmids have been found in other Acinetobacter spp.isolates able to grow on phenylpropionic acid, but in thatcase also the plasmid content was not correlated withhigh-frequency loss of the ability to grow on the aromaticsubstrate (25). Because DON2 revertants retain both theAni° phenotype and the ability to again acquire an Ani+phenotype upon selection at high aniline concentrations, a

more likely explanation for these changes is mutation orrearrangement of catabolic genes.

Aromatic catabolic pathways in A. calcoaceticus are

under strict coordinated control by substrate and metaboliteinducers and by catabolite repression (12). Initial oxidationreactions leading to catechol or protocatechuate have gen-

erally been found to be substrate induced. cis,cis-Muconatehas been shown to independently induce the next enzyme inthe catechol branch of the pathway, catechol-1,2-dioxygenase, and the enzymes which convert muconate to3-ketoadipyl coenzyme A. There are many points in thiscomplex regulatory system which may be subject to muta-tion. In addition, sequence homology in the genes codingisofunctional and consecutive enzymes in the aromatic cat-abolic pathways of Acinetobacter spp. and Pseudomonasspp. suggests a mechanism for evolution of these pathwaysthrough recombination (31). Observations of the acquisitionof an Ani+ phenotype at frequencies of 1o-4 and loss atfrequencies of 10-2 in DON isolates are commensurate withrepair and subsequent excision of genes by homologousrecombination.The nature of aniline adaptation in Acinetobacter spp. is

being examined further in this laboratory with new strainsisolated from freshwater and with Acinetobacter strainDON26. Isolates from geographically separate freshwatersites exhibit very similar aniline phenotypes to the DonRiver strains, including spontaneous loss of the Ani+ char-acter (unpublished observations). The availability of mutantswhich fail to acquire the Ani+ phenotype (DON105), as wellas other Acinetobacter strains unable to degrade aniline, willfacilitate complementation studies to isolate the anilinecatabolic pathway genes. The system illustrates the impor-tant differences in catabolic potential that can exist betweenbacteria in their natural habitat and after artificial selection.

ACKNOWLEDGMENTS

The assistance of Colleen Cotter, Alexander Salewski, and AlisonKerry in the experimental work described here is greatly appreciated.The research was supported by the Natural Science and Engineer-

ing Research Council of Canada.

LITERATURE CITED

1. Anson, J. G., and G. MacKinnon. 1984. Novel Pseudomonasplasmid involved in aniline degradation. Appl. Environ. Micro-biol. 48:868-869.

2. Aoki, K., K. Ohtsuka, R. Shinke, and H. Nishira. 1983. Isolationof aniline assimilating bacteria and physiological characteriza-tion of aniline biodegradation in Rhodococcus erythropolisAN-13. Agric. Biol. Chem. 47:2569-2575.

3. Arnow, L. E. 1937. Colorimetric determination of the compo-

nents of 3,4-dihydroxyphenylalanine-tyrosine mixtures. J. Biol.Chem. 118:531-541.

4. Bachofer, R., F. Lingens, and W. Schaefer. 1975. Conversion of

aniline into pyrocatechol by a Nocardia sp.: incorporation of

oxygen-18. FEBS Lett. 50:288-290.5. Bayly, R. C., and S. Dagley. 1969. Oxoenoic acids as metabolites

in the bacterial degradation of catechols. Biochem. J. 111:

303-307.6. Bayly, R. C., S. Dagley, and D. T. Gibson. 1966. The metabolism

of cresols by species of Pseudomonas. Biochem. J. 101:292-301.

7. Bayly, R. C., and G. J. Wigmore. 1973. Metabolism of phenoland cresols by mutants of Pseudomonas putida. J. Bacteriol.113:1112-1120.

8. Boethling, R. S., and M. Alexander. 1979. Effects of concentra-tion of organic chemicals on their biodegradation by naturalmicrobial communities. Appl. Environ. Microbiol. 37:1211-1216.

9. Boliag, J.-M., and S. Russel. 1976. Aerobic versus anaerobicmetabolism of halogenated anilines by a Paracoccus sp.Microb. Ecol. 3:65-73.

10. Bratton, A. C., E. K. Marshall, Jr., D. Babbitt, and A. R.Hendrickson. 1939. A new coupling component for sulfanil-amide determination. J. Biol. Chem. 28:537-550.

11. Button, D. K., B. R. Robertson, and K. S. Craig. 1981. Dissolvedhydrocarbons and related microflora in a fjordal seaport:sources, sinks, concentrations and kinetics. Appl. Environ.Microbiol. 42:708-719.

12. Canovas, J. L., and R. Y. Stanier. 1967. Regulation of theenzymes of the 3-ketoadipate pathway in Moraxella calco-acetica. Eur. J. Biochem. 1:289-300.

13. Davis, B. J., and L. Ornstein. 1964. Disc electrophoresis. I.Background and theory. Ann. N.Y. Acad. Sci. 121:321-404.

14. Dykhuisen, D. E., and D. L. Hartl. 1983. Selection inchemostats. Microbiol. Rev. 47:150-168.

15. Ensley, B. D., and W. R. Finnerty. 1980. Influences of growthsubstrates and oxygen on the electron transport system inAcinetobacter HO1-N. J. Bacteriol. 142:859-868.

16. Gerhardt, P., R. G. E. Murray, R. N. Costilow, E. W. Nester,W. A. Wood, N. R. Krieg, and G. B. Phillips (ed.). 1981. Manualof methods for general bacteriology. American Society forMicrobiology, Washington, D.C.

17. Haller, H. D., and R. K. Finn. 1979. Biodegradation of 3-chlorobenzoate and formation of black color in the presence andabsence of benzoate. Eur. J. Appl. Microbiol. Biotechnol.8:191-205.

18. Hansen, J. B., and R. H. Olsen. 1978. Isolation of large bacterialplasmids and characterization of the P2 incompatibility groupplasmids pMG1 and pMG5. J. Bacteriol. 135:227-238.

19. Hegeman, G. D. 1966. Synthesis of the enzymes of the mandel-ate pathway by Pseudomonas putida. J. Bacteriol. 91: 1140-1167.

20. Helm, V., and H. Reber. 1979. Investigation on the regulation ofaniline utilization in Pseudomonas multivorans. Eur. J. Appl.Microbiol. Biotechnol. 7:191-199.

21. Hintermann, G., H.-M. Fischer, R. Crameri, and R. Hutter.1981. Simple procedure for distinguishing CCC, OC, and Lforms of plasmid DNA by agarose gel electrophoresis. Plasmid5:371-373.

22. Juni, E. 1972. Interspecies transformation of Acinetobacter:genetic evidence for a ubiquitous genus. J. Bacteriol. 112:917-931.

23. Kado, C. I., and S.-T. Liu. 1981. Rapid procedure for detectionand isolation of large and small plasmids. J. Bacteriol.145:1365-1373.

24. Kaminski, U., D. Janke, H. Prauser, and W. Fritsche. 1983.Degradation of aniline and monochloroanilines by Rhodococcussp. An 117 and a pseudomonad: a comparative study. Z. Allg.Mikrobiol. 23:235-246.

25. Keil, H., U. Tittmann, and F. Lingens. 1983. Degradation ofaromatic carboxylic acids by Acinetobacter. System. Appl.Microbiol. 4:313-325.

26. Klecka, G. M., and D. T. Gibson. 1981. Inhibition of catechol2,3-dioxygenase from Pseudomonas putida by 3-chloro-catechol. Appl. Environ. Microbiol. 41:1159-1165.

27. Latorre, J., W. Reineke, and H.-J. Knackmuss. 1984. Microbialmetabolism of chloroanilines: enhanced evolution by naturalgenetic exchange. Arch. Microbiol. 140:159-165.

28. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall.1951. Protein measurement with the Folin phenol reagent. J.Biol. Chem. 193:265-275.

29. Lyons, C. D., S. Katz, and R. Bartha. 1984. Mechanisms and

788 WYNDHAM

on August 6, 2018 by guest

http://aem.asm

.org/D

ownloaded from

EVOLVED ANILINE CATABOLISM 789

pathways of aniline elimination from aquatic environments.Appl. Environ. Microbiol. 48:491-496.

30. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecularcloning: a laboratory manual, p. 104-106. Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.

31. Ornston, L. N., and W. K. Yeh. 1979. Origins of metabolicdiversity: evolutionary divergence by sequence repetition.Proc. Natl. Acad. Sci. USA 76:3996-4000.

32. Pagel, J. E., and P. L. Seyfried. 1976. Numerical taxonomy ofaquatic Acinetobacter isolates. J. Gen. Microbiol. 95:220-232.

33. Parke, D., and L. N. Ornston. 1984. Nutritional diversity ofRhizobiaceae revealed by auxanography. J. Gen. Microbiol.130:1743-1750.

34. Priefer, U. 1984. Characterization of plasmid DNA by agarosegel electrophoresis, p. 26-37. In A. Piihler and K. N. Timmis(ed.), Advanced molecular genetics. Springer-Verlag, NewYork.

35. Sharabi, N. E.-D., and L. M. Bordeleau. 1969. Biochemicaldecomposition of the herbicide N-(3,4-dichlorophenyl)-2-methylpentanamide and related compounds. Appl. Microbiol.18:369-375.

36. Spain, J. C., P. H. Pritchard, and A. W. Bourquin. 1980. Effectsof adaptation on biodegradation rates in sediment-water coresfrom estuarine and freshwater environments. Appl. Environ.Microbiol. 40:726-734.

37. Spain, J. C., P. A. van Veld, C. A. Monti, P. H. Pritchard, andC. R. Cripe. 1984. Comparison of p-nitrophenol biodegradationin field and laboratory test systems. Appl. Environ. Microbiol.

48:944-950.38. Surovtseva, E. G., and A. I. Vol'nova. 1980. Aniline as the sole

source of carbon, nitrogen and energy for Alcaligenes faecalis.Microbiologiya 49:49-53.

39. Tabak, H. H., C. W. Chambers, and P. W. Kabler. 1964.Microbial metabolism of aromatic compounds. I. Decomposi-tion of phenolic compounds and aromatic hydrocarbons byphenol-adapted bacteria. J. Bacteriol. 87:910-917.

40. Walker, N., and D. Harris. 1969. Aniline utilization by a soilpseudomonad. J. Appl. Bacteriol. 32:457-462.

41. Weatherburn, M. W. 1967. Phenolhypochlorite reaction fordetermination of ammonia. Anal. Chem. 39:971-974.

42. Wigmore, G. J., and D. W. Ribbons. 1981. Selective enrichmentof Pseudomonas spp. defective in catabolism after exposure tohalogenated substrates. J. Bacteriol. 146:920-927.

43. Wyndham, R. C. 1985. Adaptation of estuarine bacteria totoluene at low concentrations in seawater: co-metabolism oftoluene. Can. J. Microbiol. 31:910-918.

44. Wyndham, R. C., and J. W. Costerton. 1981. Heterotrophicpotentials and hydrocarbon biodegradation potentials of sedi-ment microorganisms within the Athabasca oil sands deposit.Appl. Environ. Microbiol. 41:783-790.

45. You, I.-S., and R. Bartha. 1982. Metabolism of 3,4-dichloroaniline by Pseudomonas putida. J. Agric. Food Chem.30:274-277.

46. Zeyer, J., A. Wasserfallen, and K. N. Timmis. 1985. Microbialmineralization of ring-substituted anilines through an ortho-cleavage pathway. Appl. Environ. Microbiol. 50:447-453.

VOL . 51, 1986

on August 6, 2018 by guest

http://aem.asm

.org/D

ownloaded from