Vol. 44, No. 1APPLIED AND ENVIRONMENTAL MICROBIOLOGY, July 1982, p. 246-2490099-2240/82/070246-04$02.00/0

Plasmid Involvement in Parathion Hydrolysis byPseudomonas diminuta

CUNEYT M. SERDAR,S* DAVID T. GIBSON,1 DOUGLAS M. MUNNECKE,2t AND JOHN H.LANCASTER2

Center for Applied Microbiology, Department of Microbiology, The University of Texas at Austin, Austin,Texas 78712' and Department ofBotany and Microbiology, University of Oklahoma, Norman, Oklahoma

730192

Received 23 November 1981/Accepted 30 March 1982

An organism identified as Pseudomonas diminuta was found to hydrolyzeparathion. Cells grown for 48 h contained 3,400 U of parathion hydrolase activityper liter of broth. Expression of enzymatic activity was lost at a high frequency (9to 12%) after treatment with mitomycin C. Hydrolase-negative derivatives weremissing a plasmid present in the wild-type organism. The molecular mass of thisplasmid (pCS1), as determined by electron microscopy, was about 44 x 106daltons.

The amount of pesticides produced worldwideeach year has reached over 2 x 106 tons (16), buttoxicity still remains a major problem in pesti-cide use. Parathion (PAR) is a broad-spectrumorganophosphate insecticide (for a general re-view of the literature on the degradation oforganophosphorus insecticides, see reference12).Munnecke and Hsieh (18) have studied the

degradation of PAR by a mixed microbial cul-ture and found the major metabolites to be p-nitrophenol (PNP) and diethylthiophosphoricacid (Fig. 1). Hydrolysis of PAR reduces toxici-ty by nearly 120-fold and releases water-solublemetabolites that are more available to microbialattack. PNP can be further utilized as a source ofcarbon and energy by other microorganisms (19,22, 23). Another study has shown that diethyl-thiophosphoric acid is utilized as the sole phos-phorus source (2). Enzymatic hydrolysis of PARoccurs more rapidly than does chemical hydrol-ysis by 0.1 N NaOH, and a possible industrialapplication of this enzyme has been proposed(15, 17).Involvement of naturally occurring plasmids

in degradation of the herbicides 2,4-dichloro-phenoxyacetic acid and 4-chloro-2-methylphe-noxyacetic acid has been shown (5, 21). In thispaper, we present data suggesting that a Pseudo-monas diminuta plasmid is involved in the hy-drolysis of PAR.The PAR-hydrolyzing organism was enriched

from a mixed culture as previously described(18). An organism with high PAR hydrolaseactivity was isolated and classified as P. dimin-uta by referring to the criteria listed in Bergey's

t Present address: Energy Resources Group, Cities ServiceCo., Tulsa, OK 74102.

Manual (6). This organism produced no pigmenton any of the media used for cultivation in thisstudy.PAR hydrolase activity from cells grown in

nutrient broth supplemented with 0.5% yeastextract was measured as follows. A 0.1-ml ali-quot of a whole-cell suspension was added to 5.0ml of 10 mM Tris buffer (pH 8.5) containing 10mg of PAR (obtained from Farbenfabriken Bay-er, Leverkusen, Federal Republic of Germany)per liter (34 ,uM). The production of PNP wasmeasured at 410 nm. The relationship betweengrowth of P. diminuta and production of PARhydrolase activity is shown in Fig. 2. Cell densi-ty was measured at 590 nm, and dilutions weremade when necessary to determine cell densityor PAR hydrolase activity. One unit of PARhydrolase was defined as the amount of enzymerequired to hydrolyze 1 ,mol of PAR per min.Since this organism had high PAR hydrolaseactivity, sonication of the cells was not neces-sary for enzyme detection. Production of PARhydrolase was further assayed in cultures grownon minimal salts medium (19) supplemented with0.05% yeast extract and containing 0.2% ace-tate, ethanol, succinate, histidine, or arginine asa carbon source. In all cases, PAR hydrolaseactivity was produced in the absence of addedPAR. On the basis of these results, it appearslikely that PAR hydrolase was produced consti-tutively by this culture, although the possibilitythan an inducer for the enzyme is contained inthe yeast extract cannot be excluded on thebasis of these experiments.To determine whether this enzymatic activity

was controlled by a plasmid, we attempted tocorrelate loss of PAR hydrolase activity withplasmid removal. Curing studies with mitomycinC were conducted as described by Dunn and

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S

HSC2O-PO- NO2

oC2Hs I

SI

N5C20-P-OH

i C2H5FIG. 1. Chemical structure of PAR (I) and it

drolysis products diethylthiophosphoric acid (IIPNP (III).

Gunsalus (7). After 36 h of incubationsubinhibitory concentrations of mitomyciappropriate dilutions of the organisms wereed on L-agar plates (13). We evaluated h,lase activity by selecting single coloniestransferring them into tubes containing 0.5Tris buffer (pH 8.5) with 10 ppm of PAR (Iml). Colonies with hydrolase activity rahydrolyzed PAR to PNP, as indicated bappearance of a yellow color. Of over 800nies tested, approximately 9 to 12% lost 4matic activity after treatment with 3 ,ug ofmycin C per ml. Even after cell sonicatioactivity could be detected.These cured colonies had all of the char,

istics listed for P. diminuta in Bergey's Mi(6). They differed phenotypically from theent strain only in inability to hydrolyze PA]12 antibiotics tested with Dispense-O-](Difco Laboratories), P. diminuta was resionly to streptomycin. Cured cells were

I

I

TIME (hours)

FIG. 2. P. diminuta growth and PAR hydrolaseactivity production.

resistant to streptomycin, indicating that themarker for streptomycin resistance is chromo-somal. No reversion to the hydrolase-positivephenotype was observed during repeated sub-culturing over a 2-year period. This supportsloss of a plasmid as the basis for loss of PARhydrolase activity, although induction of dele-tion mutations cannot be excluded.

Plasmid DNA was extracted from cells thathad been grown overnight in 40 ml of L-broth at30°C. Fractions enriched in plasmid contentwere prepared as described by Hansen andOlsen (9). Plasmid DNA was purified by cesiumchloride-ethidium bromide density ultracentrifu-

NO2 gation (10). Fractions containing the denser sat-ellite band were collected, extracted four times

III with cesium chloride-saturated isopropanol tots hy- remove ethidium bromide, and dialyzed againstI) and 1 liter of TES buffer (0.05 M Tris, 0.05 M NaCl,

5 mM disodium EDTA) (pH 8.0) for 12 h at 4°C.Crude and purified plasmid preparations (20 ,u)

with were subjected to agarose gel electrophoresisin C, (14) in 0.7% agarose (FMC Corp.). Electropho-nplat resis was carried out at 120 V (constant voltage)plat- for 4 h with a horizontal flat-bed slab gel appa-ydro- ratus. Gels were stained with 0.5 ,ug of ethidiumand bromide per ml of water and photographed on

ml of type 55 Polaroid film with a Polaroid MP-3 Land10iil camera equipped with a Vivitar Red Ser VIpidly filter.y the Plasmid isolation was carried out with sixcolo- hydrolase-negative colonies picked from fourenzy- curing experiments. All six clones lacked themito-* plasmid band (data not shown). Six randomlyII, HU1In, no

acter-anualpar-

R. OfDisksistantalso

tu

sc

8

FIG. 3. Agarose gel electrophoresis of plasmidDNA from P. diminuta (lanes B and C) and its curedhydrolase-negative derivative (lane D). The NAHplasmid from P. putida PpG7 (lane A) was run as astandard.

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FIG. 4. Electron micrograph of an open circular molecule of plasmid pCS1 DNA. Small molecules at thebottom, are (X174 replicative form DNA added as an internal standard. Bar = 1 ,um.

picked colonies having PAR hydrolase activitycontained plasmids. Figure 3 shows the electro-phoretic profiles of plasmid DNA from P. dimin-uta (lane C) and its cured derivative (lane D).This plasmid was designated as pCS1 in accord-ance with the recommendations of Novick et al.(20). Lane B (Fig. 3) shows pCS1 after furtherpurification by cesium chloride-ethidium bro-mide density gradient centrifugation. pCS1moved faster than the catabolic naphthalene(NAH) plasmid isolated from P. putida PpG7(ATCC 17485) (11).

Plasmid DNA was prepared for electron mi-croscopy by the aqueous technique of Davis etal. (4). The DNA was picked up on Parlodion-coated copper grids, rotary shadowed with Pt-Pd (80:20), and examined with a Hitachi HS-8electron microscope. As an internal standard,4X174 replicative-form DNA (3.558 x 106 dal-tons) (8) was used, and the molecular mass ofpCS1 was obtained from measurements of sevenopen circular molecules. Figure 4 shows a mole-cule of pCS1 in open circular form. The averagemolecular mass was calculated to be 43.9 (±0.6)x 106 daltons.

In this report, we have shown that after treat-ment with mitomycin C, P. diminuta lost theability to hydrolyze PAR and that a plasmid was

also eliminated. These results suggest that PARhydrolase activity may be mediated by plasmidDNA. Attempts to demonstrate transformationor conjugal transfer of pCS1 into a cured strainof P. diminuta were not successful. However,this could have been due to the low solubility ofPAR in the agar media used to select for trans-formants or transconjugants, which would inter-fere with recognition of the PAR hydrolase-positive phenotype.

Little information on the enzymatic systemsinvolved in complete degradation of PAR isavailable. The initial reaction in PNP utilizationis catalyzed by a monooxygenase in a Moraxellasp. (23). An Escherichia coli phosphoesterasethat hydrolyzes PAR has been described (24).Plasmid-encoded enzymes in Alcaligenes spp.(5) are involved in 2,4-dichlorophenoxyaceticacid degradation. Dagley (3) has suggested thatthis compound is the type of herbicide whichwould be designed for biodegradation if themetabolic pathway were understood, since thefinal products are normal cell metabolites. Ananalogous situation is observed in PAR metabo-lism, in which hydrolysis of the phosphotriesterbond yields metabolites that can be further uti-lized (2, 19, 22, 23).

Production ofPAR hydrolase by the P. dimin-

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uta strain tested appears to be correlated withthe presence of an autonomous plasmid andconstitutive. This plasmid, which, as determinedby contour length measurement, has an estimat-ed molecular mass of 44 x 106 daltons, presum-ably carries the hydrolase gene. Attempts totransform the plasmid back into cured strainshave not been successful; thus, the possibilitythat mitomycin C in some way inactivated agenetic element carried in the chromosome can-not be excluded as a mechanism for loss ofhydrolase activity upon mitomycin C treatment.However, the fact that no strains carrying theplasmid were hydrolase negative and no strainslacking the plasmid were hydrolase positivestrongly suggests that the PAR hydrolase gene iscarried on plasmid pCS1. The ability of this P.diminuta strain to hydrolyze PAR may be due toa mutation in a nonspecific esterase. A casesimilar to this was demonstrated in Pseudomo-nas aeruginosa studies in which a single aminoacid change has been shown to affect the speci-ficity of wild-type acetamidase, enabling theenzyme to act on acetanilide (1).

We thank I. C. Gunsalus for the gift of P. putida PpG7,D. T. Brown for helpful suggestions regarding electron mi-croscopy, and Roberta DeAngelis for typing the manuscript.

This work was supported in part by grant F-440 from theRobert A. Welch Foundation.

LITERATURE CITED

1. Brown, P. R., and P. H. Clarke. 1972. Amino acid substi-tution in an amidase produced by an acetanilide-utilizingmutant of Pseudomonas aeruginosa. J. Gen. Microbiol.70:287-298.

2. Cook, A. M., C. G. Daughton, and M. Alexander. 1978.Phosphorus-containing pesticide breakdown products:quantitative utilization as phosphorous sources by bacte-ria. Appi. Environ. Microbiol. 36:668-672.

3. Dagley, S. 1972. Degradation of synthetic organic mole-cules in the biosphere, p. 345. National Academy ofSciences, Washington, D.C.

4. Dais, R. W., M. Simon, and N. Davidson. 1971. Electronmicroscope heteroduplex methods for mapping regions ofbase sequence homology in nucleic acids. Methods Enzy-mol. 21:413-427.

5. Don, R. H., and J. M. Pemberton. 1981. Properties of sixpesticide degradation plasmids isolated from Alcaligenesparadoxus and Alcaligenes eutrophus. J. Bacteriol.145:681-686.

6. Doudoroff, M., and N. J. PaHleroni. 1974. In R. E. Bu-chanan and N. E. Gibbons (ed.), Bergey's manual of

determinative bacteriology, 8th ed., p. 236. The Williams& Wilkins Co., Baltimore.

7. Dunn, N. W., and I. C. Gunsalus. 1973. Transmissibleplasmid coding early enzymes of naphthalene oxidation inPseudomonas putida. J. Bacteriol. 114:974-979.

8. Fisher, H. W., and R. C. Williams. 1979. Electron micro-scopic visualization of nucleic acids and of their complex-es with proteins. Annu. Rev. Biochem. 48:649-679.

9. Hansen, J. B., and R. H. Olsen. 1978. Isolation of largebacterial plasmids and characterization of the P2 incom-patibility group plasmids pMG1 and pMG5. J. Bacteriol.135:227-238.

10. Humphreys, G. O., G. A. Willshaw, and E. S. Anderson.1975. A simple method for the preparation of largequantities of pure plasmid DNA. Biochim. Biophys. Acta383:457-463.

11. Johnston, J. B., and I. C. Gunsalus. 1977. Isolation ofmetabolic plasmid DNA from Pseudomonas putida. Bio-chem. Biophys. Res. Commun. 75:13-19.

12. Laveglia, J., and P. A. Dahm. 1977. Degradation of or-ganophosphorous and carbamate insecticides in the soiland by soil microorganisms. Annu. Rev. Entomol.22:483-513.

13. Lennox, E. S. 1955. Transduction of linked genetic char-acters of the host by bacteriophage P1. Virology 1:190-206.

14. Meyers, J. A., D. Sanchez, L. P. Elwell, and S. Falkow.1976. Simple agarose gel electrophoretic method for theidentification and characterization of plasmid deoxyribo-nucleic acid. J. Bacteriol. 127:1529-1537.

15. Munnecke, D. M. 1976. Enzymatic hydrolysis of organo-phosphate insecticides, a possible pesticide disposalmethod. Appl. Environ. Microbiol. 32:7-13.

16. Munnecke, D. M. 1978. Detoxification of pesticides-usingsoluble or immobilized enzymes. Process Biochem.13:16-19.

17. Munnecke, D. M. 1980. Enzymatic detoxification of wasteorganophosphate pesticides. J. Agric. Food Chem.28:105-111.

18. Munnecke, D. M., and D. P. H. Hsieh. 1974. Microbialdecontamination of parathion and p-nitrophenol in aque-ous media. Appi. Microbiol. 28:212-217.

19. Munnecke, D. M., and D. P. H. Hsieh. 1976. Pathways ofmicrobial metabolism of parathion. AppI. Environ. Micro-biol. 31:63-69.

20. Novick, R. P., R. C. Clowes, S. N. Cohen, R. Curtiss III,N. Datta, and S. Falkow. 1976. Uniform nomenclature forbacterial plasmids: a proposal. Bacteriol. Rev. 49:168-189.

21. Pemberton, J. M., and P. R. Fisher. 1977. 2,4-D Plasmidsand persistence. Nature (London) 278:732-733.

22. Simpson, J. R., and W. C. Evans. 1953. The metabolismof nitrophenols by certain bacteria. Biochem. J. 55:xxiv.

23. Spain, J. C., 0. Wyss, and D. T. Gibson. 1979. Enzymaticoxidation ofp-nitrophenol. Biochem. Biophys. Res. Com-mun. 88:634-641.

24. Zech, R., and K. D. Wigand. 1975. Organophosphate-detoxicating enzymes in E. coli. Gel filtration and isoe,lc-tric focusing of DFPase, paraoxonase and unspecificphosphohydrolases. Experientia 31:157-158.

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