Plasmid Specifying Total Degradation of 3-Chlorobenzoate by a

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Plasmid Specifying Total Degradation of 3-Chlorobenzoate by a Modified ortho Pathway DEB K. CHATTERJEE, S. T. KELLOGG, SUZANNE HAMADA, AND A. M. CHAKRABARTY* Department ofMicrobiology and Immunology, University ofIUinois, Medical Center, Chicago, IUlinois 60612 Received 9 September 1980/Accepted 27 February 1981

A p id, terned pAC25, specifying biodegradation of 3-chlorobenzoate in a strain of Pseudomonas putida has been characterized. During growth of the plasmid-harboring cells with 3-chlorobenzoate, there was an accumulation of 3chlorocatechol and fl-chloromuconic acid as intermediates and release of more than 80% of the chlorine in the form of inorganic chloride. The plasmid had a mean molecular mass of 68 x 106 daltons and was transmissible to a number of Pseudomonas species such as P. aeruginosa, P. putida strain PpG1, and P. putida strain PRS1. Transfer of pAC25 to various catechol-negative mutants of P. putida strain PRS1 showed that the chromosomally coded pyrocatechase was not complemented by the plasmid-specified pyrocatechase, which appeared to be specific for the chlorinated catechols. In contrast to benzoate, which was metabolized by the ortho pathway through ,B-ketoadipate as an intermediate, the plasmid specified ortho cleavage of the the chlorocatechols through maleylacetate as an intermediate.

Chlorobenzoates are a group of compounds that occur in the environment in large amounts either because of their release as herbicides (16, 22) or as products of cometabolism of the polychlorinated biphenyls by mixed or pure cultures (14). Degradation of chlorobenzoates by mixed or pure cultures has been described (1, 6, 18). In particular, Knackmuss and his associates (6, 7, 15, 23) have described the isolation and characterization of a Pseudomonas species, B13, capable of total degradation of 3-chlorobenzoate (3Cba) by elaborating ortho pathway enzymes specific for chlorocatechol metabolism. It appears that completely new enzymes, specific for chlorobenzoate degradation, have evolved in nature due to the widespread release of this and other synthetic haloaromatic compounds. It is, however, also possible that such enzymes reflect a preadaptive variant of a pathway that has evolved under other selective presures and that is coincidentally capable of attacking 3Cba. There is no report in the literature regarding the status of the genes encoding chlorobenzoate biodegradation in Pseudomonas B13 or other microorganims. In this report, we describe the characterization of a transmissible plasmid that specifies a complete 3Cba-biodegradative pathway in a strain of P. putida. A preliminary account of this work has been published (4).

was isolated from sewage samples in Niskayuna, N.Y. Other P. putida and P. aeruginosa strains have been described elsewhere (3, 13). The composition of the synthetic minimal medium used for propagation of the strains (PAS medium) is given below ("Isolation of plasmid DNA"). The genotypic characteristics and the plasmids harbored by these strains are given in Table 1. Measurements. UV and visible spectra of the supernatant fluid were taken after taking equal portions from the growth medium at various times, centrifuging the celLs, diluting the samples, and scanning them in a Cary model 15 spectrophotometer. Total phenols were determined by the method of Folin and Ciocalteu (12). 1,2-Dihydroxyphenols were determined by the method of Evans (9). Chloride ion concentrations were determined with an ion-selective combination chloride electrode (model IS-146, Laza Research Laboratories, Los Angeles, Calif.), calibrated initially with NaCl. ,Ketoadipate was determined by the color reaction as described by Rothera (25). Curing and conjugational tansfer of plasmids. The methods for curing the 3Cba-degradative plasmids were the same as described previously (3). The methods used for conjugational transfer of the plasmid have been described (13). Synthesis of Mae. Maleylacetic acid (Mac) was synthesized according to Eisner et al. (8), using maleic anhydride as a starting material. Briefly, the steps involved were: maleic anhydride -. methyl hydrogen fumarate -* trans-f8-carbomethoxyacrylyl chloride -* ethyl-trans-,8-carbomethoxyacrylylmalonate -- Mac

MATERIALS AND METHODS Organisms and media. The strain of P. putida capable of utilizing 3Cba as the sole source of carbon

Cells were grown in a Isolation of plasmid synthetic medium (pH 7.0) containing (in grams per liter): K2HPO4.3H20, 2.96; KH2PO4, 0.87; NH4Cl, 1.1; MgSO4, 0.097; MnSO4. H20, 0.025; FeSO4. 7H20, 0.005;

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acid). (trans-dihydro-f6-ketomuconic DNA.

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TABLE 1. List of bacterial strains Plasnid Genotype

Derivation or reference P. putida Wild type AC858 pAC25 4 Wild type AC859 Spontaneous ade-i AC860 pAC25 NGa treatment of AC858 AC861 Wild type Mitomycin C AC862 trp-6 NG treatment of AC859 P. putida PpGl met-i AC10 13 Wild type AC30 13 met-i AC863 pAC25 Conjugation (AC860 x AC10) Wild type AC864 pAC25 Conjugation (AC860 x AC30) cba-3 DF3 pAC25 NG treatment of AC864 DF7 cba-7 pAC25 NG treatment of AC864 cba-ii DF11 pAC25 NG treatment of AC864 Wild type DF17 Curing (spontaneous) Wild type DF21 pAC25 Transduction P. putida PRSl his-i AC105 M. L. Wheelis AC79 catAb L. N. Ornston PRS2015 catB L. N. Ornston catC PRS2191 L. N. Ornston catD PRS5 L. N. Ornston P. aeruginosa PAO AC590 trp-54 res-i str-6 mod-i B. W. Holloway AC865 trp-54 res-i str-6 mod-I pAC25 Conjugation (AC869 x AC590) AC48 his-101 This laboratory NG, N-methyl-N-nitro-N-nitrosoguanidine. b The catA (catechol 1,2-oxygenase-negative), catB (MLE-negative), catC (muconolactone isomerase-negative), and catD (enol lactone hydrolase-negative) mutants are phenotypically Ben- and were kindly given to us by L. N. Ornston (see reference 21 for properties of such mutants). Strain designation

CaCl2.2H20, 0.0015; and L-ascorbic acid, 0.005. SubRESULTS strate (chlorobenzoates) was added at a concentration of 1 mg/ml. Biochemistry of 3Cba degradation. We Usually cells from a 1-liter culture were used for the have previously reported the of a P. isolation of plamid DNA. Cells were harvested at putida strain from sewage thatisolation can utilize 3Cba mid- or late exponential phase, washed with 250 ml of 50 mM Tris-chloride, pH 8.0, and resuspended in the as the sole source of carbon with a doubling time same buffer at a ratio of 1 g/5 ml. The plasmid DNA of about 170 min (4). Growth of this strain was isolated by the method of Casse et al. (2). The (AC858) with 3Cba in a mineral medium gave plasmid DNA was purified by cesium chloride-ethid- positive Folin-Ciocalteu tests for phenolic subium bromide equilibrium density gradient centrifuga- stances and, during the exponential phase of tion in a fixed-angle type 50 rotor at 40,000 rpm at growth, a positive Evans test (9) for the presence 20°C for 48 h. The refractive index of the solution was of 1,2-dihydroxyphenolic compounds. Examinaadjusted to 1.3993 (CsCI used at a concentration of 1 tion of the fluids of the growth g/ml). The plasmid band, visualized by a UV transil- medium for supernatant characteristic absorption spectra luminator, was collected from the bottom with the demonstrates that during the early phase of help of a Beckman gradient collector. Ethidium bromide was removed by extraction with isopropanol growth (early log phase), there is a shift in saturated with 3 M sodium chloride and 0.3 M sodium absorption from a 284-nm peak of 3Cba to a 275citrate buffer. Cesium chloride was removed by di- to 276-nm peak of 3-chlorocatechol (10, 24). alysis against 10 mM Tris-chloride containing 1 mM With increasing accumulation, the A,,. shifts to EDTA, pH 8.0. 268 to 270 nm, suggesting considerable accumuAgarose gel electrophoresis. The agarose gel lation of 3-chlorodihydroxybenzoate (24). Durelectrophoresis was done with 0.7% agarose, using 36 ing late log phase and early stationary phase, mM Tris-chloride, 30 mM NaH2PO4, and 1 mM the absorption maximum shifts to 264 nm, sugEDTA, pH 7.8, as the gel and electrode buffer. The gel was electrophoresed for 6 h at a 120-mA gesting accumulation of 8-chloromuconic acid constant current. The gel was stained with the elec- (10). Accumulation of all of these internediates trode buffer contapning 1 ,ug of ethidium bromide per follows the basic pattern of chlorocatechol meml for 20 to 25 min and visualized by a UV transillu- tabolism by other Pseudomonas strains grown minator. with 4-chlorophenoxyacetate (4Cpa) or 3Cba,

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where 4Cpa or 3Cba is known to be converted to chloromuconic acid before cleavage of the aromatic ring. Based on previous observations (10, 23), the 3Cba+ strain AC858 is believed to harbor a similar degradative pathway involving chlorocatechol and chloromuconic acids as intermediates (Fig. 1). The release of chloride as a function of growth of strain AC858 with 3Cba as the sole carbon source can be seen from the results in Fig. 2. At the end of the log phase (48 h), about 80% of the 3Cba chlorine was released as inorganic chloride into the medium. Curing, transmissibility, and other characteristics of the 3Cba-degradative plasmid. Species of Pseudomonas and other bacterial genera are known to harbor, on plasmids, the genes specifying biodegradation of a number of naturally occurring hydrocarbons as well as synthetic compounds (11). Since the status of the genes governing chlorocatechol metabolism in various bacteria (10, 18, 23) is unknown, we were interested in determining if the genes controlling 3Cba metabolism in P. putida AC858 may be borne on a plasmid. The 3Cba-positive character in AC858 was found to be unstable, 3Cba- segregants being observed regularly at a frequency of 0.3 to 0.35%. To determine whether the putative 3Cba-degradative plasmid was conjugative or nonconjugative, the transmissibility of the 3Cba-degradative property to the 3Cba-negative cells as well as other P. putida and P. aeruginosa cells was studied. An Ade- mutant of the 3Cba+ P. putida (AC860) was used as a donor, and one of the spontaneously derived 3Cba- (AC859) as well as several other Pseudomonas strains were

L

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4.5

0.6

3.0

0.4 . .

C.

x 0.2

II

20

Time(hr)

30

40

FIG. 2. Growth and release of chloride ions during metabolism of 3Cba by strain AC858. The cells were grown in minimal medium containing 7.5 mM 3Cba. Similar results were obtained with pAC25+ exconjugants such as AC864 or AC865. Symbols: (A) growth; (0) Cl- release.

used as recipients. To avoid problems of restriction, a restriction-negative mutant of P. aeruginosa (AC590) was also used as a recipient. The transfer frequency was about 50-fold higher in the restriction-negative mutant than in the other strains, but in general all Pseudomonas species accepted the plasmid at a low frequency (Table 2). There was no chromosomal mobilization from the donor to any of the recipients. This conjugative plasmid is henceforth designated pAC25. The biochemistry and enzymology of chlorobenzoate degradation have been extensively studied by Knackmuss and his colleagues (7, 23). The growth of Pseudomonas B13 on 3Cba has been demonstrated to elicit production of isoenzymes specific for chlorocatechol metabolism (7). The usual ortho pathway enzymes are only active in using chlorinated catechols as weakly I C°°N ~ ~~~ 'COcoN substrates, and the bulk of the chlorocatechol is metabolized by the isoenzymes (15, 23). The cli Ci isoenzymes appear to be specific for chlorinated 4-Chiolrscat,cho -. 'T -CarbxJmothyiom Is substrates and are not active toward the nonA'- botewlide chlorinated parents. A similar situation is observed with pAC25+ cells, since transfer of pAC25 to Ben- mutants allows good growth with 3Cba but not with benzoate, even in the presence COON -COO O N iir~ of inducing quantities of 3Cba (4). To determine TC how many of the pAC25-coded enzymes may eycle,~~ICCN2 n ro also be active toward nonchlorinated catechol or I its metabolites, we transferred pAC25 to a numON P-Nyiroymuc.oic Maleylacetic ber of catechol-negative mutants of P. putida FIG. 1. Proposedpathway for the metabolism of 4- strain PRS1 (20, 21). pAC25-coded enzymes chlorocatechol, according to Evans et al. (10). An could not restore the missing enzymatic funcidentical pathway is believed to be operative in the tions in catA, catC, and catD mutants, but could metabolism of 3-chloro-, 4-chloro-, and 3,5-dichloro- do so in catB mutants (Table 3). Loss of pAC25 catechols by other Pseudomonas species (see refer- from such catB/pAC25 exconjugants renders ence 15). TCA, Tricarboxylic acid. them Ben-, suggesting that the Ben' phenotype ON

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acid

COON

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acid

acid

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CHATTERJEE ET AL.J.BTEOL

TABLE, 2. Transmissibility of the 3Cba-degradative property to a number of Pseudomonas specie89, with AC860 as donor' Recipient Select Transfer fre- Exconjugant phenoA089 AC861 AC862 AC862 AC30 AC10 AC10

3Cba+ 3Cba+ 3Cba+

Tfrp+

3Cba+ 3Cba+ Met+ 3Cba+ His+ 3Cba+ His+ 3Cba+

2 x 10O6 1 8

X 10-6 X 10-7

2 x 10per infected cell) with class C mutants (two mutants). No appreciable number of transductants was obtained when recipients within each group were infected with phages grown on individual members of the group. Phages grown on class B mutants produced transductants at a frequency of nearly 1 x 10-7 to 3 x 10-7 for class A mutants and about 8 x 10-7 to 1 x 106 for class C mutants. Phages grown on class C similarly produced a large number of trnsductants (average frequency of 1 x 106) with class A recipients and a smaller number (frequency of nearly 1 x 10-7) with class B mutants. To determine whether each class of mutants could utilize Mac, one representative mutant from each group was inoculated into minimal Mac medium and grown for 4 days (Fig. 3). Whereas mutant DF3 (class A) and mutant DF7 (class B) grew with Mac as the sole source of carbon, mutant DF11 (class C) grew extremely slowly with it. Thus, mutants DF3 and DF7 appeared to be 3Cba- Mac', whereas mutant DF11 appeared to be 3Cba- Mac. To determine whether the 3Cba- and Macphenotypes in DF11 were due to a single mutation or to two separate mutations, we checked the growth of a 3Cba+ transductant of DF11 (DF21) with Mac. The transductant (DF21) regained its ability to utilize Mac, whereas a plasmid-negative variant was incapable of growing with it (Fig. 3). It therefore appeared that the 3Cba- Mac- phenotypes were due to a single mutation; i.e., similar to 4Cpa+ pseudomonads, Mac- mutations made the cell phenotypically 3Cba-, suggesting Mac as an intermediate of 3Cba degradation. None of the three class of mutants could be transduced to 3Cba+ with phages grown on the plasmidless strain (DF17). Thus, the pAC25- strain P. putida PpG1 did not

14

48

Growth plriolihr)

71

96

FIG. 3. Growth of 3Cba- mutants of pAC25' P. putida PpGl, pAC25 PpGl, and a 3Cba+ transductant with Mac as the only source of carbon. The cells were inoculated into minimal medium with 1 mg of Mac per ml and grown under shaking at 30°C. Symbols: (A) DF3; (0) DF7; (0) DF11; (0) DF21; (A) DF17 (pAC25 PpGI).

appear to harbor any functional 3Cba-degradative genes. Size of the pAC25 plasmid. pAC25 is very difficult to isolate by the usual methods of plasmid DNA isolation. Initial attempts to isolate the plamid DNA from P. putida or P. aeruginosa celLs, grown with or without 3Cba as the sole source of carbon, invariably met with failure. With the new method described by Casse et al. (2) after some modification, it is now possible to isolate the plasmid DNA with a low yield. After electrophoresis on a 0.7% agarose gel, pAC25 had essentially the same mobility as pAC30 (68 megadaltons [Mdal]), but moved faster than TOL (75 Mdal) and slower than SAL (49 Mdal) (Fig. 4a). Electron microscope contour length determination of a large number (64 molecules) of covalently closed circular forms of the plasmid demonstrated the size to be about 68 ± 2.2 Mdal. Covalently closed circular forms of identical size have been isolated from both pAC25+ P. aeruginosa and pAC25+ P. putida cells, but not from the pAC25- parents, confirming the mean size of pAC25 to be about 68 Mdal. Figure 4b shows the EcoRI digestion pattern of these four plasmids. pAC25 demonstrated the appearance of 11 fragments after EcoRI digestion, and the sum total of these fragments was about 69 Mdal. Incompatibility characteristics. To see whether pAC25 may be incompatible with other

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FIG. 4. (a) Electrophoretic mobility of SAL, pAC30, pAC25, and TOL plasmid DNAs on 0.7% agarose gel; (b) electrophoretic mobilities offragments of these plasmids after digestion with EcoRI.

hydrocarbon-degradative plasmids, we introduced plasmids of P1, P2, and P9 incompatibility groups such as RP4-TOL (pAC8), CAM, SAL, and TOL into pAC25+ P. putida cells. pAC25 was compatible with pAC8 and CAM, but showed interesting behavior with TOL and SAL. Since both TOL and SAL belong to the P9 inc group (17), we expected that pAC25 would be compatible either with both or with none. Instead, pAC25 was found to be compatible with SAL but apparently incompatible with TOL. When pAC25 was introduced into TOL+ cells, only 2 of 22 were pAC25+ TOL+, the rest (20) having lost TOL. The two TOL+ pAC25+ colonies were found to be unstable, segregating either TOL or pAC25, depending upon selection. The problem of coexistence of TOL and pAC25 appears to relate to metabolic regulatory problems rather than a problem of incompatibility or replication. Reineke and Knackmuss (23) have shown that 3Cba, when acted upon by the meta pathway enzymes induced in the presence of the TOL plasid, produces a chlorinated meta pathway intermediate which is extremely toxic for the cells. 3Cba also irreversibly inactivates catechol 2,3-dioxygenase, the first enzyme of the meta pathway, thereby making the cells phenotypically TOL- (23). Growth in the presence of 3Cba therefore invariably leads to a loss of the TOL plasnid or inactivation of the plasmidspecified catechol 2,3-oxygenase, making the pathway nonfunctional. DISCUSSION In this study, we have not examined the biochemistry and enzymology of the 3Cba-degradative pathway, other than the appearance of dihydroxyphenolic compounds by colorimetric means and the probable production of chlorocatechol and chloromuconic acids by spectroscopic scanning. The major attempts have in-

stead been directed toward identifying Mac as an intermediate of 3Cba biodegradation by mutant isolation and correlation of the abilities of these mutants to utilize 3Cba and Mac. The involvement of these intermediates in the utilization of 3Cba by pAC25+ cells must therefore be considered tentative and relies on detailed studies on the biochemistry of the pathway by previous workers (10, 23, 26). Although we have not directly demonstrated that the enzymes allowing utilization of 3Cba are indeed coded by pAC25, the alternative possibility, that these genes are chromosomal in the 3Cba- strains P. putida PpGl and PRS1 and in P. aeruginosa PAO but are somehow turned on in the presence of a positive-acting element coded by pAC25, seems uinlikely. Transductional experiments have demonstrated that the plasmidless PpGl strain does not have any functional 3Cba-degradative genes. Similar to other Pseudomonas degradative plasmids, it is likely that pAC25 codes for a complete pathway for the degradation of 3Cba. The plasmid nature of the genes coding for 3Cba degradation is apparent from the observed curability, transferability to a large number of Pseudomonas species under conditions where there is no chromosomal mobilization, and the fact that covalently closed circular molecules of a mean molecular mass of 68 Mdal have been isolated from the 3Cba+ cells but not from the 3Cba- segregants. Plasmids, of course, are known to specify biodegradation of various hydrocarbons and chlorinated compounds such as p-chlorobiphenyl and 2,4-dichlorophenoxyacetic acid (11). A plsid that encodes biodegradation of a synthetic nylon oligomer such as 6-aminohexanoic acid cyclic dimer by a Flavobacterium species has also been described recently (19). The interesting aspect about the evolution of a plasmid such as pAC25 is that the plasmiid en-

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codes genes that allow dechlorination from the chlorinated compound. Previously described plasmids for p-chlorobiphenyl and 2,4-dichlorophenoxyacetic acid degradation do not harbor genes that allow dechlorination of these compounds (4). The ability of the plamid pAC25 to complement catB mutants appears to indicate that the plasmid-specified enzyme is functional also toward the nonchlorinated substrate, whereas the pyrocatechase produced by Pseudomonas B13 (7) as well as the one coded by pAC25 plasmid appears to be specific for chlorinated catechol and does not act on the nonchlorinated parent. A similar situation has been described by Crawford et al. (5) in a strain of Bacillus brevis that can utilize 5-chlorosalicylate as a sole source of carbon and energy. This bacterium synthesizes a specific 5-chlorosalicylate 1,2-dioxygenase, a novel enzyme that cleaves the aromatic ring while it is substituted with only one hydroxyl group. This enzyme cannot use salicylate as a substrate, and a halogen other than iodine or a methoxyl group is required at the 5 position for a substituted salicylate to be a substrate for the dioxygenase. Crawford et al. (5) concluded that the loss of the halogen substituent as halide ion from ring carbon 5 appears to be enzyme mediated, although the substrate specificity of this enzyme could not be studied because of the unavailability of suitable substrates. In case of the pAC25-coded dehalogenation step, it is not clear whether there is a separate dechlorinase enzyme catalyzing dechlorination and the subsequent lactonization of the muconic acid product by the plasmidspecified MLE or whether the chloromuconic acid produced from chlorocatechol is unstable enough to spontaneously release chloride before being converted to the lactone. The evidence that the lactone produced from chloromuconic acid is not metabolized via f,ketoadipate is quite strong, since the cells accumulated large quantities of fi-ketoadipate when grown with benzoate, but at undetectable levels when grown with 3Cba. Also, pAC25-positive 3Cba- Mac- mutants as well as plaidless (pAC25-) cells could not metabolize Mac, whereas both pAC25+ cells and 3Cba+ transductants of 3Cba- Mac- mutants grew with Mac as the sole carbon source. The plasmid therefore appears to code for a set of ortho enzymes that is different from the ,8-ketoadipate pathway enzymes, except perhaps for MLE. It would be interesting to determine the genetic homology between the plasmid-coded MLE and the MLE of the chromosomally coded ,B-ketoadipate pathway, as well as their enzymatic characteristics. The occurrence of a Mac pathway for the degradation of 4Cpa (10), 3Cba by Pseudomonas

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B13 (26), and 3Cba by pAC25-coded enzymes raises the interesting question regarding the evolutionary relationships among the degradative genes present in these bacteria, all ofwhich were isolated in different countries at different times. Although the status of the genes for the biodegradation of 4Cpa is unknown, there is evidence that a plamid may govern the biodegradation of 3Cba by Pseudomonas B13 (K. Timmis, personal communication). It would be interesting to compare the genetic homology between the 3Cba-degradative plasmid in Pseudomonas B13 and pAC25 to determine whether these two plasmids have a common ancestry. ACKNOWLEDGMENTS This investigation was supported by grants PCM 79-17526 and PFR 79-05499 from the National Science Foundation. LITERATURE CITED 1. Boliag, J. M. 1974. Microbiol transformation of pesticides. Adv. Appl. Microbiol. 18:75-130. 2. Casm, F., C. Boucher, J. S. Julliot, ML Michel, and J. Denarie. 1979. Identification and characterization of large plasmids in Rhizobium meliloti using agrose gel electrophoresis. J. Gen. Microbiol. 113:229-242. 3. Chakrabarty, A. M. 1972. Genetic basis of the biodegradation of salicylate in Pseudomonas. J. Bacteriol. 112:815-823. 4. Chakrabarty, A. M. 1980. Plasmids and dissimilation of synthetic enviromnental pollutants, p. 21-30. In C. Stuttard and K. R. Rozee (ed.), Plasmids and transposons: environmental effects and maintenance mechanisms. Academic Press, Inc., New York. 5. Crawford, R. L., P. E. Olson, and T. D. Frick. 1979. Catabolism of 5-chlorosalicylate by a Bacills isolated from the Mississppi river. Appl. Environ. Microbiol.

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cyclohexadiene-1,2-diol-1-carboxylic acid by a mutant strain ofAlcaligenes eutrophus. Biochemistry 10:25302536. 25. Rothera, A. C. H. 1908. Note on the sodium nitro-prusside reaction for acetone. J. Physiol. 248:491494. 26. Schmidt, E., and H.-J. Knacnkmua. 1980. Chemical structure and biodegradability of halogenated aromatic compounds: conversion of chlorinated muconic acids into maleoylacetic acid. Biochem. J. 192:339-347.