APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1993, p. 1898-1903 Vol. 59, No. 60099-2240/93/061898-06$02.00/0Copyright ©D 1993, American Society for Microbiology

5-Hydroxyquinoline-2-Carboxylic Acid, a Dead-EndMetabolite from the Bacterial Oxidation of

5-Aminonaphthalene-2-Sulfonic AcidBERND NORTEMANN,1 ANDREA GLASSER,1 REINHARD MACHINEK,2 GERD REMBERG,2

AND HANS-JOACHIM KNACKMUSS3*Technische Chemie und Chemische Verfahrenstechnik, Universitat-GH Paderborn, 4790 Paderborn, 1 Institutfur Organische Chemie, Universitat Gottingen, 34 Gottingen,2 and Institut fur Mikrobiologie der Universitat

Stuttgart, Azenbergstrasse 18, 7 Stuttgart 1,3 Germany

Received 24 August 1992/Accepted 30 March 1993

5-Aminonaphthalene-2-sulfonate (5A2NS) is converted by strain BN6 into 5-hydroxyquinoline-2-carboxylate(5H2QC). The authenticity of this new compound is confirmed by nuclear magnetic resonance and massspectrometry. Its formation is explained by a spontaneous cyclization of the hypothetical metabolite6'-amino-2'-hydroxybenzalpyruvate. The formation of 5H2QC as a dead-end product of 5A2NS preventsNADH regeneration so that 5A2NS oxidation is limited by the internal NADH pool.

Strain BN6 isolated from a bacterial mixed culture degrad-ing 6-aminonaphthalene-2-sulfate (6A2NS) (15) oxidizes abroad spectrum of amino- and hydroxynaphthalene-2-sul-fonates. The catabolic pathway is analogous to that de-scribed for the degradation of naphthalene-2-sulfonate byPseudomonas testosteroni A3 (3, 12). In contrast to thelatter organism, strain BN6 lacks salicylate-hydroxylatingactivities. Consequently, naphthalene-2-sulfonates with OHor NH2 substituents in position 5, 6, 7, or 8 are metabolizedto the corresponding hydroxy- or aminosalicylates in equi-molar amounts. An exception is 5-aminonaphthalene-2-sul-fonate (5A2NS), which is not converted into 6-aminosalicy-late as the major product (15).The present work describes the isolation of the metabolite

from SA2NS and its identification as 5-hydroxyquinoline-2-carboxylate (5H2QC).

MATERIALS AND METHODS

Chemicals. Naphthalene-2-sulfonic acid, 5A2NS, and6A2NS were a gift from Bayer AG, Leverkusen, Germany.Quinoline-2-carboxylic acid and 5-hydroxyquinoline werepurchased from Aldrich GmbH & Co. KG, Steinheim,Germany. All other chemicals used for mineral salts mediaand the buffer solution were obtained from E. Merck AG,Darmstadt, Germany, or from Fluka, Buchs, Switzerland.

Microorganisms. Isolation and characterization of the6A2NS-degrading culture have been published previously(15). Strain BN6 isolated from this mixed culture is depos-ited as Pseudomonas vesicularis DSM 6383 at the GermanCollection of Microorganisms and Cell Cultures. Recently,however, it has been identified as a member of the alphasubclass of proteobacteria with the misnamed Chromobac-terium folium NCTC 10591 as the next phylogenetic neigh-bor (5).Media and growth conditions. Media were prepared in a

mineral base containing (per liter) the following: 5.5 g ofNa2HPO4, 0.5 g of (NH4)2SO4, 0.2 g of MgSO4. 7H20, 50mg of Ca(NO3)2 4H20, 10 mg of ammonium ferric citrate,and 1 ml of a trace solution described by Pfennig and Lippert

* Corresponding author.

(16) without iron salt and EDTA. The mineral salt mediumwas supplemented with a given carbon source and auto-claved at 121°C.

Cells were grown in 100- to 3,000-ml Erlenmeyer flaskscontaining, at most, 20% medium. Cultures were incubatedat 30°C on a rotary shaker (type RC 106; Infors AG, Basel,Switzerland) at 120 rpm.Maintenance of strain BN6. During growth of strain BN6

with 6A2NS in axenic culture, 5-aminosalicylate is accumu-lated as a metabolite and then is subject to rapid autoxida-tion. For the maintenance of strain BN6, a 5-aminosalicy-late-degrading strain such as strain BN9 (15, 18) wascocultivated as separate streaks parallel to those of strainBN6. This avoided accumulation of 5-aminosalicylate andallowed subcultivation of both strains under selective con-ditions.

Conversion of5A2NS. Cells of strain BN6 were grown witha mixture of glucose (15 mM) and naphthalene-2-sulfonate(0.5 mM) as an inducer. The mixed bacterial culture (15) wasgrown with 6A2NS (10 mM) as the sole source of carbon andenergy. Organisms were harvested in the late exponentialgrowth phase, washed, and suspended in phosphate buffer(50 mM, pH 7.3). The cell suspensions were shaken for 30min before SA2NS was added.

Protein estimation. Irrespective of the growth substrateused, a linear correlation existed between the protein con-tent of exponentially growing cells of strain BN6 (10 mMglucose or 5 mM 4-hydroxybenzoate) or the mixed culture(10 mM 6A2NS) and the optical density (OD). An OD at 546nm (OD546) of 1.0 corresponded to a protein content of 0.11to 0.13 mg/ml (14, 17). Therefore, the protein content ofwhole cells was estimated by measuring the OD546 with aspectrophotometer (type UV-240; Shimadzu GmbH, Dussel-dorf, Germany). If the OD546 exceeded a value of 0.3,samples of the culture fluid were appropriately diluted withphosphate buffer (50 mM).

Analytical methods. Naphthalenesulfonates and metabo-lites were analyzed by reverse-phase high-pressure liquidchromatography (HPLC 655-11 liquid chromatograph[Merck, Darmstadt, Germany] equipped with an automaticinjector [WISP 710 B; Millipore GmbH, Waters Chromatog-raphie, Eschborn, Germany]). The eluate was examined

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with a UV detector (Spectroflow SF 770; Schoeffel, West-wood, N.Y.). Signals were analyzed by use of a computingintegrator (Autolab System 1; Spectra-Physics, Santa Clara,Calif.). A hyperchrome column SC 125 (internal diameter,125 by 4.6 mm; Bischoff, Leonberg, Germany) filled withLiChrospher RP8 (5-pLm particles, Merck) was used toseparate individual compounds. Detection limit at 215 nm formost aromatic compounds including 5H2QC was approxi-mately S p,mol. liter-'.The mobile phase contained 0.1% (wt/wt) H3PO4 in dis-

tilled water with different amounts of methanol (20 to 60%[wt/wt]) depending on the polarity of the substrate or metab-olite. Samples (5 pl) of culture fluids were injected after cellshad been removed by centrifugation in 1-ml MicroTest tubesfor 3 min at 20,000 rpm (Mikro-Haemokrit; Heraeus Christ,Osterode, Germany).Ammonia was determined with glutamate dehydrogenase

by the method of Da Fonseca-Wollheim et al. (7). Thereaction mixture was slightly modified and contained, in afinal volume of 1 ml, 50 ,umol of imidazole (pH 7.9), 10 ,umolof 2-oxoglutarate, 1 ,umol of EDTA, 2 ,umol of ADP, 0.14,umol of NADH, and 0.01 ml of glutamate dehydrogenase inglycerol (Boehringer GmbH, Mannheim, Germany). Thereaction was started by the addition of 5 to 25 ,ul of cell-freeculture fluid or standard solution of ammonia. The decreasein NADH concentration was measured at 366 nm (e = 3.4mM-' cm-').Mass spectra of the isolated metabolite were measured by

use of a Varian MAT 311-A mass spectrometer (Varian,MAT, Bremen, Germany). Nuclear magnetic resonance(NMR) spectra were recorded with a Varian XL200 (1HNMR spectra) or a Varian VXR200 (13C NMR spectra)instrument (Varian, Palo Alto, Calif.). Tetramethylsilanewas used as an internal standard.

RESULTS

Turnover of5A2NS. Resting cells of strain BN6, which hadbeen grown with glucose (15 mM) in the presence of naph-thalene-2-sulfonate (0.5 mM), metabolized 5A2NS with aninitial rate of 60 to 186 p.mol- min-' g of protein-'. Am-monia was not released during 5A2NS turnover. As indi-cated by HPLC analysis, consumption of the substrate wasaccompanied by the accumulation of three metabolites.Their retention times (R,) were 210, 300, and 460 s (mobilephase, 30% methanol, 70% H20, 0.1% H3PO4). The increasein intensity of the chromatographic signals for these prod-ucts always corresponded to the amount of substrate con-sumed. However, the intensities of the chromatographicsignals at 210 and 460 s were less than 5% of that observedat 300 s (measured at 215 nm). The metabolite which elutedafter 300 s appeared to be the major product and, therefore,was primarily investigated.

After separation by HPLC, fractions containing the5A2NS oxidation product (R, 300 s) were evaporated at30°C. The UV spectra measured in methanol-H20 (30:70)exhibited a Xmax = 248 nm at pH 7.5 and km. = 262 nm atpH 1.5 or pH 11.0. These data could not be correlated withthose described for 6-aminosalicylate (Xm,. = 234, hydro-chloride in methanol) by Hillis and Gould (10). Since strainBN6 exhibited no salicylate hydroxylase activity, the prod-uct from 5A2NS oxidation could not be a metabolite of6-aminosalicylate.When resting cells of strain BN6 were incubated with

5A2NS, turnover activity was lost before complete con-sumption of the substrate (Fig. 1). If naphthalene-2-sulfonate

TABLE 1. Mass spectrometry fragmentation pattern of 5H2QC

mle % Intensity of Interpretationbasal signal

189 100 M+

145 69 M+ -C02144 36 M+ -COOH116 24 (M+ - COOH) - CO89 28 [(M+- COOH) - CO] -CHN

was added to this reaction mixture, it was metabolizedinstantaneously with a high rate, yielding stoichiometricamounts of salicylate. Moreover, some activity for SA2NSoxidation was restored by this treatment. Oxidative activitycould also be restored by addition of glucose (Fig. 2).Under the given conditions, the maximum concentration

of SA2NS which could be metabolized was approximately 5mM even if the concentration of biomass was increased(OD546 ' 20) or cosubstrates were added to the reactionmixture in large excess. When inactivated cells were har-vested by centrifugation (15 min at 5,000 rpm) and resus-pended in fresh phosphate buffer, only some of the activityfor the turnover of 5A2NS could be restored by the additionof cosubstrates.

Isolation of the major product of 5A2NS. HPLC studiesindicated that with either an axenic culture of strain BN6 ora 6A2NS-degrading mixed culture the major product fromSA2NS oxidation was obtained with almost the same yield.Since it was difficult to induce naphthalenesulfonate-con-verting activity in the axenic culture of strain BN6, oxidationwas carried out by the mixed culture. This could easily begrown under selective and inducing conditions with 6A2NSas the sole source of carbon and energy. Washed cells of themixed community (OD546 20) were incubated with 5A2NS(4 mM). When the consumption of the substrate came to ahalt, cells were removed from the culture fluid by centrifu-gation. The pellet was then resuspended in the same quantityof fresh phosphate buffer. Notably, this suspension alsocontained some of the major product (5 to 10% of the totalamount prior to centrifugation). Obviously, this compoundwas poorly soluble in aqueous solution so that part of itprecipitated in the culture fluid and then was redissolved infresh buffer.The metabolite (Re, 300 s) could not be extracted with

organic solvents such as diethylether, n-hexane, ethylace-tate, or chloroform at pH values between 1.5 and 8.0. Since,compared with other components of the culture medium, theproduct from 5A2NS oxidation appeared to be poorly solu-ble in water, it should precipitate first during evaporation.Therefore, the cell-free culture fluid was evaporated at 30°Cto a small volume, and the precipitate was harvested bycentrifugation. The dark pellet obtained was resuspended inthe same volume of distilled water and shaken for 10 min.This procedure was repeated twice. Finally, the precipitateexhibited a greyish-yellow color. It was resuspended inmethanol and shaken for 10 min. Undissolved particles wereremoved by centrifugation. On acidification, the color of thesupernatant (pH 1.5 to 3.0) changed from brown to brightyellow. The acidified solution was evaporated, and solidresidue was dried over phosphorus pentoxide. The yellowishmaterial obtained consisted of small platelets exhibiting aweak metallic lustre.A small portion of the yellowish product was dissolved in

phosphate buffer and analyzed by HPLC. A single chro-

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1900 NORTEMANN ET AL.

2.0

Ec0o4-

0c0

1.5 .

1.0 .

Q5

0

0 60 120 180

time (min)FIG. 1. Effects of naphthalene-2-sulfonate on the rate of cooxi-

dation of 5A2NS. Cells of strain BN6 were grown with glucose (15mM) in the presence of naphthalene-2-sulfonate (0.5 mM), har-vested, resuspended (0.30 mg of protein per ml) in phosphate buffer(50 mM, pH 7.2), and shaken without substrate. After 30 min,5A2NS was added and incubation continued. When consumption of5A2NS came to a halt, naphthalene-2-sulfonate (2NS) was added,yielding salicylate. The accumulation of the product from 5A2NSoxidation is not figured.

25

201

E

cnz

LO

C

0

coCu

a)00

1.5

1.01

as!-

0

U 100 200

time (min)FIG. 2. Effects of glucose on SA2NS turnover. Resting cells (0.8

mg of protein per ml) of strain BN6 (40-ml suspension in 50 mMphosphate buffer, pH 7.2) were incubated with SA2NS (2.5 mM).After addition of a second part of SA2NS, consumption ratedecreased. At this time (indicated by an arrow), half of the mixturewas transferred into an Erlenmeyer flask containing 20 mg ofglucose (approximately 6 mM). Consumption of SA2NS withoutadditional substrate (0) or in the presence of glucose (-) was

measured by HPLC. Accumulation of the metabolite of SA2NS isnot figured.

TABLE 2. 'H NMR data of 5H2QC'

Protonb Chemical Descriptionshift (ppm)1 8.04 AB system (J,2 = 8.65 Hz)2 8.67 AB system (J2,1 = 8.65 Hz); long-range

coupling 2,55 not resolved3 7.06 ABC system (J3,4 = 8.47 Hz, J3,5 = 1.22 Hz)4 7.63 ABC system (143 = 8.47 Hz,14,5 = 7.25 Hz)5 7.65 ABC system (J54 = 7.25 Hz, J53 = 1.22 Hz)

a The compound was dissolved in dimethyl sulfoxide-d6, and the spectrumwas recorded at 200 MHz.

b Notation of protons is given in Fig. 3.

FIG. 3. Formula of SH2QC and notation of protons given inTable 2.

matographic signal was observed which, by retention timeand in situ-measured UV spectrum, was identical to thecorresponding signal of the major metabolite prior to isola-tion. Moreover, the product did not contain detectableamounts of 5A2NS or other metabolites.

Identification of 5H2QC. Mass spectrometric analysis ofthe product gave a molecular mass of 189.06, correspondingto the structural formula C1OH7NO3. By comparison of thefragmentation pattern (Table 1) with those of quinoline-2-carboxylic acid, 5-hydroxyquinoline, and 4-hydroxyquino-line-2-carboxylic acid (not illustrated) the 5A2NS oxidationproduct appeared to be a hydroxyquinoline-2-carboxylicacid. This was supported by high-resolution measurementsof the molecular ion and of those of other key fragments.The proton magnetic resonance data (Fig. 3; Table 2) of

the 5A2NS oxidation product were consistent with 5H2QC.The protons 1 and 2 of the aromatic C-3 C-4 form an ABsystem (doublets at 8 = 8.04 or 8.65, respectively) with aspin coupling constant of J12 = 8.65 Hz. The signals for thedoublet at 8.67 ppm (proton of C-4) are broader than those at8.04 ppm, indicating a nonresolved long-distance couplingwith proton 5 of C-8. An ABC system (8H-3 = 7.06, 8H-4 =7.63, SH-5 = 7.65) is formed by the protons of C-6, C-7, andC-8.

Extensive 13C NMR studies (Table 3; Fig. 4 and 5) andcomparison of the NMR data observed with those of quino-line, quinoline-2-carboxylic acid, and 5-hydroxyquinolineclearly confirmed that the metabolite from 5A2NS turnoverwas 5H2QC. The signal at 8 = 166.33 results from the carbonatom of the carboxyl group. The chemical shift of C-5 is153.20 for 5H2QC and 153.10 for 5-hydroxyquinoline. Thesignals for C-2 are 8 = 148.64 for 5H2QC and 8 = 149.00 forquinoline-2-carboxylic acid. Multiplicity of the signals wasdetermined by a multiplett selection experiment (APT pulsesequence) and demonstrated that the carbon atoms with 8 =

5A2NS

2NS

I"

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5H2QC, A DEAD-END METABOLITE OF 5A2NS 1901

TABLE 3. '3C NMR data of 5H2QCa

C atomb Chemical Status ofshift (ppm) protonationc

1 166.3 Nonprotonated2 148.6 Nonprotonated3 119.2 Protonated4 132.2 Protonated5 153.2 Nonprotonated6 110.1 Protonated7 130.8 Protonated8 120.0 Protonated9 147.9 Nonprotonated10 120.6 Nonprotonated

a The compound was dissolved in dimethyl sulfoxide-d6, and the spectrumwas recorded at 50.3 MHz.

b Notations as given in Fig. 4.Multiplicity of the signals were determined by a multiplett selection

experiment.

1COOH

3 2

4\ N

HO *

6 7

FIG. 4. Notation of C atoms of 5H2QC as given in Table 3.

F I (PPM)

9.0

8.8 4

8.6 (if8.4

7.8 7 87.6

7. 4

7.2

7.0

6.8

120.59, 147.86, 148.64, and 153.20 are not protonated. Asshown by a carbon-proton correlation experiment (HET-COR), C-3 and C-4 are correlated with the protons of the ABsystem, and C-6, C-7, and C-8 are correlated with theprotons of the ABC system (Fig. 5).

Yield and purity of 5H2QC. As described above, a singlechromatographic signal was observed when SH2QC wasanalyzed by HPLC. Mass spectra and all NMR spectra werefree from signals other than those for SH2QC. Therefore, itwas concluded that the product did not contain significantamounts of organic impurities. However, elementary analy-sis revealed only 60% of the calculated values for nitrogenand carbon. From this, the estimated molecular extinctioncoefficients would be 19.0 mM-1 cm-' at Xma = 248 nm inaqueous solution (pH 7.5), 20.0 mM-' cm-' at Xm. = 258nm in aqueous solution (pH 1.5), and 19.0 mM-1 cm-' at aXmax = 260 nm in methanol with 0.1% HCI. In acidicsolutions, two additional signals at 315 and 325 nm wereobserved. However, the intensities of these signals were lessthan 5% of that measured at 258 nm.Assuming that the purity of 5H2QC was only 60%, ap-

proximately 0.75 mmol of 5H2QC per mmol of 5A2NSoxidized was accumulated in the culture fluid. By loss duringisolation procedure, 780 mg (2.48 mmol) of 5H2QC wasobtained per g of 5A2NS (4.48 mmol).

Further purification of 5H2QC was hampered by its poorsolubility in all solvents tested. From boiling ethanol, abright-yellow and partially crystalline matter was obtained.Elementary analysis revealed 62.23% C (calculated forC1OH7NO3: 63.49%), 6.65% N (calculated 7.41%), and 4.02%H (calculated 3.70).

F2 (PPM)

FIG. 5. 13C-_H correlated NMR spectrum.

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I

OH ID

H2N

COOH

FIG. 6. Proposed catabolic pathway for 5A2NS. Dotted arrowsindicate that minor amounts of the substrate are funnelled into thisroute.

Cooxidation of 6-ethoxy-5A2NS. Resting cells of strainBN6 also metabolized 6-ethoxy-SA2NS (2 mM) at a rate of80 ,umol- min-' g of protein-'. This was approximately75% of the turnover rate with 5A2NS in a control experi-ment.HPLC analysis indicated the accumulation of a single

product. The intensity of its signal always corresponded tothe amount of substrate consumed. The product exhibitedcharacteristics similar to those of SH2QC. Particularly, thebathochromic shift in the UV spectrum from km. = 259 nmat pH 7.5 to km. = 278 nm at pH 2.0 and the poor solubilitycorresponded to the properties of 5H2QC. Since purificationof this metabolite proved to be even more difficult than thatof 5H2QC, the product from 6-ethoxy-5A2NS oxidation wasnot further analyzed.

DISCUSSION

In contrast to the catabolism of 6A2NS and 7- or 8-ami-nonaphthalene-2-sulfonate and 5-, 6-, 7-, or 8-hydroxynaph-thalene-2-sulfonate, 5A2NS was not converted into thecorresponding salicylates by strain BN6 (15). Moreover,cells readily lost their catabolic activity with 5A2NS as asubstrate. This indicated that, for some reasons, the degra-

dation stops at a catabolic step prior to the formation of6-aminosalicylate.The product from 5A2NS turnover could not be extracted

with organic solvents and appeared to be poorly soluble inwater. Therefore, the product was isolated by precipitationfrom the concentrated culture supernatant. The molecularformula of this compound was established as C10H7NO3 byhigh-resolution mass spectrometry and could not be relateddirectly to one of the expected metabolites from 5A2NS. Bycomparison with the mass fragmentation pattern of quino-line-2-carboxylic acid, 5-hydroxyquinoline, and 4-hy-droxyquinoline-2-carboxylic acid, the isolated metaboliteappeared to be a hydroxyquinoline-2-carboxylic acid. It wasclearly identified as 5H2QC by extensive NMR studies. Theformation of this new compound can readily be explained byan intramolecular condensation reaction of 6'-amino-2'-hy-droxybenzalpyruvate (compounds IVa and IVb in Fig. 6).Obviously, this primary metabolite is subject to rapid cy-clization before it can be cleaved into pyruvate and 6-ami-nosalicylaldehyde by the 2'-hydroxybenzalpyruvate-aldo-lase. Since 5-hydroxynaphthalene-2-sulfonate, a structuralanalog of 5A2NS, was quantitatively converted into thecorresponding salicylate, catabolic misrouting of 5A2NSmust be due to the NH2 group.Naphthalene dioxygenases are multicomponent enzyme

systems which are NAD(P)H dependent (6, 9, 11, 13).NADH-regenerating reactions during catabolism of naphtha-lene-2-sulfonate by strain BN6 are the assimilation of thepyruvate from C-1, C-2, and C-3 and the oxidation ofsalicylaldehydes by salicylaldehyde dehydrogenase. Duringoxidation of 5A2NS, however, the formation of 5H2QCprevents NADH regeneration, so that the cells readily loseactivity. Significantly, part of the activity was restored bythe addition of cosubstrates such as naphthalene-2-sulfonateor glucose. When the concentration of 5H2QC in the culturefluid exceeded 4 mM, however, consumption of 5A2NScame to a halt even in the presence of additional substratesor at higher concentrations of biomass (OD.46 . 20). Inaddition to NADH depletion, cells appear to be inactivatedby 5H2QC.Known hydroxyquinoline-2-carboxylic acids are 4- and

8-hydroxyquinoline-2-carboxylic acids (kynurenic and xan-thurenic acids) as metabolites of tryptophan (1, 2, 4). It hasbeen reported for both acids and their salts that they tend tocontain crystal water which is only released at temperaturesof >140'C. Like 5H2QC, kynurenic and xanthurenic acidexhibit low solubilities in water and most organic solvents.The UV spectral characteristics of 5H2QC, including thered-shift as the pH is lowered from 7.5 to approximately 1.5,resemble those of quinoline-2-carboxylic acid and 4- or8-hydroxyquinoline-2-carboxylic acid, as described by El-Ezaby et al. (8). This is due to protonation of the nitrogenwith probable formation of a zwitterion. The latter is stabi-lized by intramolecular hydrogen bonding with the acidgroup. From this, it can be explained that SH2QC cannot beextracted from the culture fluid with organic solvents.Although the purity of 5H2QC on the basis of elementary

analysis was only about 60%, it obviously did not containsignificant amounts of organic impurities. This was shown byHPLC, mass spectrum, and NMR spectra. Therefore, it ismost likely that the compound was isolated as a salt (e.g., ahydrochloride) and contained water of crystallization. Asdemonstrated by a turnover experiment with 6-ethoxy-5A2NS, other 5H2QCs with additional substituents appearto be accessible from the corresponding 5A2NSs.As exemplified by hydroxy- and aminonaphthalene sul-

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fonates, complex chemical structures harboring high xeno-biotic character may be subject to partial degradation ratherthan mineralization. In Pseudomonas vesicularis BN6, in-complete degradation of naphthylsulfonates is due first of allto the lack of any salicylate-oxidizing activity giving rise tosalicylates as dead-end metabolites. In the case of 5A2NS,prior to the formation of the corresponding salicylate, ahighly reactive intermediate which stabilizes itself and es-capes further degradation by intramolecular condensation isgenerated. By following the catabolic sequence of naphtha-lene, most naphthylsulfonates liberate pyruvate from C-1,C-2, and C-3. This is utilized for growth of strain BN6,provided that toxic salicylates were removed from the me-dium by a second organism harboring complementary cata-bolic pathways. In contrast, 5A2NS metabolism is totallyunproductive or, because of the loss of energy (reductionequivalents), even counterproductive.

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