for Degradation of 2,4-Dichlorophenoxyacetic Acid by Plasmid pJP4

Vol. 171, No. 6

JOURNAL OF BACTERIOLOGY, June 1989, p. 3385-3390

0021-9193/89/063385-06$02.00/0 Copyright C) 1989, American Society for Microbiology

Recruitment of a Chromosomally Encoded Maleylacetate Reductase for Degradation of 2,4-Dichlorophenoxyacetic Acid by Plasmid pJP4 JEROME J. KUKOR,1 RONALD H. OLSEN,'* AND JUNE-SANG SIAK2 Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, Michigan 481090620,1 and Biomedical Science Department, General Motors Research Laboratories, Warren, Michigan 48090-90552

Received 28 November 1988/Accepted 20 March 1989

When Pseudomonas aeruginosa PAOlc or P. putida PPO200 or PPO300 carry plasmid pJP4, which encodes for the degradation of 2,4-dichlorophenoxyacetic acid (TFD) to 2-chloromaleylacetate, cells do not TFD and UV-absorbing material with spectral characteristics of chloromaleylacetate accumulates in the culture medium. Using plasmid pRO1727, we cloned from the chromosome of a nonfluorescent pseudomonad, Pseudomonas sp. strain PKO1, 6- and 0.5-kilobase BamHI DNA fragments which contain the gene for maleylacetate reductase. When carrying either of the recombinant plasmids, pRO1944 or pRO1945, together with pJP4, cells of P. aeruginosa or P. putida were able to utilize TFD as a sole carbon source for growth. A novel polypeptide with an estimated molecular weight of 18,000 was detected in cell extracts of P. aeruginosa carrying either plasmid pRO1944 or plasmid pRO1945. Maleylacetate reductase activity was induced in cells of P. aeruginosa or P. putida carrying plasmid pRO1945, as well as in cells of Pseudomonas strain PKO1, when grown on L-tyrosine, suggesting that the tyrosine catabolic pathway might be the source from which maleylacetate reductase is recruited for the degradation of TFD in pJP4-bearing cells of Pseudomonas sp. strain PKO1. enzymes grow on

Chlorinated aromatic compounds have been used extensively during the past 40 years in both manufacturing and agriculture. Although many of these compounds show environmental persistence, the chlorophenoxy herbicides 2methyl-4-chlorophenoxyacetic acid and 2,4-dichlorophenoxyacetic acid (TFD) appear to be readily degraded by soil and aquatic bacteria (2, 24, 33). Of the various bacterial strains that have been isolated for their ability to degrade TFD, the most intensively studied have been those identified as Alcaligenes eutrophus (8, 28, 29). A. eutrophus JMP134 carries an 80-kilobase (kb), broad-host-range P1 incompatibility group plasmid, designated pJP4, which encodes genes essential for the catabolism of TFD (9, 10). Plasmid pJP4 has also been transferred to several other strains of gramnegative bacteria, and in such transconjugants growth on TFD as the sole carbon source has been reported for A. eutrophus, A. paradoxus, Pseudomonas putida (8), P. oxalaticus (13), P. cepacia, P. pickettii, and Pseudomonas sp. strain PKO1 (this study). Transposon mutagenesis, DNA cloning, and DNA sequence analysis have been used to localize the catabolic functions encoded by pJP4, and it has been shown that the plasmid encodes enzymes for the conversion of TFD to 2-chloromaleylacetate (CMA) (1, 10, 16, 32). However, enzymes for the further conversion of CMA have never been shown to be present on plasmid pJP4. Further metabolism of CMA to tricarboxylic acid cycle intermediates apparently depends on enzymes specified by chromosomal genes in A. eutrophus (10). Our laboratory has recently isolated a derivative of plasmid pJP4, designated pRO103, which is a Tnl721-bearing deletion mutant of the parent plasmid that constitutively expresses the TFD degradation genes (18). Plasmid pRO103 confers a TFD-positive phenotype on various pseudomonal strains, including Pseudomonas sp. strain PKO1, a strain recently isolated from soil. However, we have noted that strains of P. aeruginosa and P. putida are unable to grow on *

TFD as the sole carbon source when carrying pRO103, even though such cells express the TFD degradation genes (18). Cultures of TFD-grown P. aeruginosa or P. putida carrying pRO103 accumulate UV-absorbing material with spectral characteristics of CMA (12). This fact suggested to us that P. aeruginosa and P. putida lack enzymes for the catabolism of CMA, which is produced from TFD degradation by pRO103encoded enzymes. In this report, we describe the molecular cloning and characterization of DNA fragments, obtained from Pseudomonas sp. strain PKO1, that encode a maleylacetate (MAA) reductase. When present in pRO103-bearing strains of P. aeruginosa or P. putida, the cloned MAA reductase gene (mar) renders these strains capable of growth on TFD as the sole carbon source. Additionally, we present evidence that MAA reductase is induced by tyrosine (Tyr) in Pseudomonas sp. strain PKO1, as well as in a Pseudomonas sp. strain PKO1 DNA fragment cloned into P. aeruginosa and P. putida. We also show that chloride is released stoichiometrically from dichlorinated catechols by cells carrying both the cloned mar gene and pRO103. MATERIALS AND METHODS Bacterial strains and plasmids. The strains and plasmids used in this study are listed in Table 1. Plasmid pRO1946 was derived by cloning a BamHI fragment from plasmid pJP4 into the unique BamHI site within the tetracycline resistance gene of vector plasmid pRO1727. Don et al. (10) had previously shown that the genes for the degradation of chlorocatechol through CMA were located on the BamHI C fragment of pJP4. Media and growth conditions. Minimal medium (VBG or MMO) and complex medium (TN) xyere prepared as described previously (7, 27). Tetracycline was used in selective media at 25 ,ug/ml for all strains except P. aeruginosa, for which it was used at 50 ,ug/ml. Ticarcillin was used at 250 p.g/ml for P. aerulginosa and at 500 p.g/ml for P. putida. When grown for enzyme assays, bacteria were cultured in

Corresponding author. 3385

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Strain or plasmid

Strains Alcaligenes eutrophus AE0106 Pseudomonas sp. strain PKO1 Pseudomonas aeruginosa PAO1c Pseudomonas cepacia DBO1 PC383 TL249 Pseudomonas pickettli PKO2 Pseudomonas putida pp0200b PPO300

TABLE 1. Bacterial strains and plasmids. Relevant characteristic(s) or marker(s)"

Reference or source

Prototroph, TfdPrototroph Prototroph

JMP134 cured of pJP4 (18) This study 19

Prototroph Prototroph Prototroph Prototroph

Laboratory stock D. Chatterjee (17) T. Lessie (23) ATCC 27511

Prototroph Prototroph

35 ATCC 17514

Plasmids pRO103 pRO1727 pRO1944 pRO1945 pRO1955 pRO1946

Constitutive mutant of pRO101, Tfd' Hgr Tcr 18 Tir Tcr 7 TirMaa+ This study This study Tir Maa+ Tir Maa+, Sall subclone of pRO1945 This study Tir, 12-kb BamHI C fragment of pJP4 This study a Abbreviations: Hg', Tir, and Tc', resistance to mercury, ticarcillin, and tetracycline, respectively; Tfd-, TFD negative; Tfd+, TFD positive; Maa+, MAA

reductase positive. b p. putida PPO200 is a derivative of strain mt-2 (ATCC 33015) that has been cured by us of its TOL plasmid.

100 ml of MMO medium with aeration and supplemented with carbon substrates to a final concentration of 10 mM. TFD was added to MMO medium to a final concentration of 0.05%, and Casamino Acids (CAA), when used as a supplemental carbon source, were added to a final concentration of 0.3%. P. aeruginosa and P. cepacia were grown at 37°C; all other strains were grown at 30°C. Genetic techniques. Isolation of chromosomal and plasmid DNAs, restriction endonuclease digestion and molecular cloning, and transformation and plasmid matings were carried out as described previously (3, 7, 19, 20, 25, 26). Measurement of enzyme activity. Cells were grown for enzyme assays to a density that gave an apparent A425 of 1.0 to 1.5 (Spectronic 21 spectrophotometer; Bausch & Lomb, Inc., Rochester, N.Y.) and were harvested by centrifugation at 10,000 x g for 15 min. The cell pellets were washed twice in 50 mM sodium-potassium phosphate buffer (pH 6.8) containing 1 mM dithiothreitol, 0.1 mM EDTA, and 1 ,uM flavin adenine dinucleotide, and the cells were disrupted sonically by four 15-s 200-W bursts with a Braun-Sonic 1510 apparatus. Cellular debris was removed by centrifugation at 100,000 x g and 5°C for 1 h, and the clear supernatant solution was used immediately for enzyme assays. MAA reductase activity was assayed by measuring the decrease in the A340 with NADPH as the cosubstrate (5, 6, 14, 15). The reaction was carried out at 30°C in 1.0-ml quartz cuvettes with a 1-cm light path. The final volume of 1.0 ml contained 885 pI of 50 mM sodium-potassium phosphate buffer (pH 6.8), 10 ,u1 of 10 mM NADPH, 5 RIt of 10 mM MAA, and 100 ,ul of appropriately diluted cell extract. Under these conditions, 1 enzyme unit corresponds to an absorbance decrease of 6.1 optical units per min (14). Protein was determined by the method of Bradford (4) with bovine serum albumin as the standard. Enzyme specific activities are reported as micromoles of substrate or cosubstrate utilized per minute per milligram of protein. MAA was prepared by the procedure of Reineke and Knackmuss (30), except that the starting substrate was 4-chlorocatechol (4CC), which was enzymatically converted to chloromuconate by a purified chlorocatechol-1,2-dioxy-

genase obtained from the cloned HindIll G fragment of plasmid pRO103 (J. J. Kukor and R. H. Olsen, unpublished data). UV absorbance spectra were measured on a Shimadzu UV-160 spectrophotometer. Electrophoretic methods. Total soluble cellular proteins, precipitated with ammonium sulfate at 40 to 60% saturation, were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) by the method of Laemmli (22). A model SE250 vertical slab gel unit (Hoefer Scientific Instruments, San Francisco, Calif.) was used for all separations. Samples were boiled for 3 min in solubilization buffer (60 mM dithiothreitol, 1% SDS, 0.44 mM phenylmethylsulfonyl fluoride, 10% glycerol, and 0.04% bromphenol blue in 10 mM Tris [pH 6.8]). Gels were run for 15 min at 25 mA through a 4% acrylamide stacking gel and for a further 60 min at 20 mA through either a 12% or a 15% acrylamide separating gel. For visualization of proteins, the gels were stained with Coomassie blue. Chloride release. Cells of P. putida PP0300 carrying plasmid pRO103 and grown in MMO medium containing 0.05% TFD and 0.3% CAA were broken by sonic oscillation, and cell extracts were prepared as described above. A suitable amount of the cell extract (usually 0.05 to 0.15 mg of total protein) was added to 10 ml of 50 mM sodiumpotassium phosphate buffer (pH 6.8) containing 0.01 mM 4CC or 4,5-dichlorocatechol (45DCC). Reaction mixtures were incubated at room temperature for at least twice the time required for the complete utilization of 45DCC. Chloride concentrations were measured with a model 9617B combination chloride electrode (Orion Research, Inc., Cambridge, Mass.). Chemicals and reagents. All of the chemicals, enzymes, and reagents used in these studies were of the highest purity commercially available. Enzymes and reagents used for DNA manipulations were purchased from International Biotechnologies, Inc., New Haven, Conn., Bethesda Research Laboratories, Inc., Gaithersburg, Md., or Boehringer Mannheim Biochemicals, Indianapolis, Ind., and were used as suggested by the suppliers. 4CC and 45DCC were obtained from Helix Biotech Ltd., Richmond, British Columbia,

MALEYLACETATE REDUCTASE RECRUITMENT

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M

z z

1

2

3387

3

pRO1944 MAR

I

,,I

I . I 11

I

-

mmmlF--j

I

pRO1945

MAR

FIG. 2. SDS-PAGE profile of 40 to 60% saturation ammonium sulfate fractions of soluble cellular proteins of P. aeruginosa carry-

ing pRO1944 (lane 1), pRO1945 (lane 2), or cloning vector pRO1727 (lane 3). Molecular weight markers (lane M) were bovine albumin (66,000), ovalbumin (45,000), glyceraldehyde-3-phosphate dehydrogenase (36,000), carbonic anhydrase (29,000), trypsinogen (24,000), trypsin inhibitor (20,100), and oi-lactalbumin (14,200). The arrow indicates the position of the putative MAA reductase.

mm

pR01955 MAR

I

0

I

1

i

2

I

3

I

4

I

5

I

6

kb

FIG. 1. Restriction maps of the mar-containing clones derived

from Pseudomonas sp. strain PKO1. The positions of relevant restriction sites from the plasmid cloning vector are shown flanking the cloned chromosomal DNA fragments. The approximate location of the mar gene is shown.

Canada. Disodium ticarcillin was from Beecham Laboratories, Bristol, Tenn. Vitamin-free CAA, as well as all other bacteriological medium components, were purchased from Difco Laboratories, Detroit, Mich. Tetracycline, sodium pyruvate (PYR), and Tyr were obtained from Sigma Chemical Co., St. Louis, Mo. RESULTS Cloning of chromosomal DNA fragments from Pseudomonas sp. strain PKO1 carrying the mar gene required for growth on TFD. Chromosomal DNA from Pseudomonas sp. strain PKO1 was digested with restriction endonuclease BamHI and ligated into the unique BamHI site within the tetracycline resistance gene of vector plasmid pRO1727 as described previously (26). This ligation mixture was used to transform cells of P. aeruginosa PAO1c carrying pRO103, a TnJ721-bearing derivative of plasmid pJP4. Transformants were initially selected on TN medium containing both tetracycline and ticarcillin and were then tested for growth on TFD minimal medium. TFD-positive transformants were screened for plasmid content by a rapid alkaline lysis procedure. Results of such a screening revealed, in addition to pRO103, two recombinant plasmids that were larger than the pRO1727 cloning vector. After purification of these plasmid DNAs in cesium chloride-ethidium bromide gradients, restriction digest analysis demonstrated that the larger recombinant plasmid, designated pRO1945, contained a 6-kb BamHI insert, whereas the smaller recombinant plasmid, designated pRO1944, contained a 0.5-kb BamHI insert. Plasmids pRO1944 and pRO1945 were mapped with restriction endonucleases (Fig. 1). To localize the mar gene on pRO1945, we made a series of Sall deletion subclones. One of these deletion subclones,

designated pRO1955, which contained the 0.5-kb SallBamHI termini (map coordinates 0 to 0.5 kb and 5.5 to 6.0 kb [Fig. 1]) of pRO1945, was found to encode a functional MAA reductase (data not shown). Comparative restriction endonuclease mapping of pRO1955 and pRO1944 demonstrated that the two plasmids have a similar distribution of restriction endonuclease sites in the mar-containing region of the cloned fragments (Fig. 1). Detection of the mar gene product. Plasmid-encoded proteins from pRO1944 and pRO1945 (and cloning vector pRO1727 as a control) were synthesized in P. aeruginosa PAO1c grown on 0.3% CAA. From results described below, it had been determined that growth on this carbon source would result in adequate induction of MAA reductase activity. Electrophoresis of total crude cell lysates on polyacrylamide gels resulted in poor separation of proteins, and the amount of mar gene product synthesized was too low to be detected in Coomassie blue-stained gels (data not shown). To maximize the detectability of the mar gene product, we fractionated total crude cell lysates with ammonium sulfate. The 40 to 60% ammonium sulfate fraction, which contained all of the MAA reductase activity, was separated on an SDS-PAGE gel (Fig. 2) and stained with Coomassie blue. A band with an apparent Mr of 18,000 was present in the ammonium sulfate fraction from cells carrying pRO1944 or pRO1945 but was not present in a comparable fraction from cells carrying cloning vector pRO1727. Expression and regulation of the cloned mar gene. MAA reductase activity was detected in TFD-CAA- or TFDPYR-grown cells of P. aeruginosa PAO1c carrying pRO1944 or pRO1945 (Table 2). Plasmid-free cells of P. aeruginosa or cells carrying only pROlO3 had no detectable MAA reductase activity. CAA-grown cells of P. aeruginosa carrying either pRO1944 or pRO1945 also exhibited MAA reductase activity. A similar result occurred when cells were grown on Tyr, a component of CAA. PYR-grown cells of P. aerdginosa, on the other hand, had no detectable MAA reductase activity when pRO1945 was present; however, cells carrying pRO1944 had fully induced levels of enzyme activity. The same pattern of induction of MAA reductase activity was obtained in cells of P. putida PP0300 carrying the cloned mar gene on pRO1944 or pRO1945 as in P. aeruginosa PAO1c cells carrying these plasmids (Table 3). These results suggest that a negative regulatory gene that encodes a repressor and that is present on plasmid pRO1945 has been deleted from plasmid pRO1944.

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TABLE 2. Activity of MAA reductase cloned from Pseudomonas sp. strain PKO1 in P. aeruginosa PAOI

TABLE 4. Activity of MAA reductase in various bacterial strains carrying pRO103 Activity' on: CAA

Tyr

PYR

PYR

CA

Ty

PY

0.003 0.990 0.001 0.833 0.004 0.567 0.002 0.683 0.001 0.603 0.003 0.944 0.001 0.001 0.001 0.002 0.001 0.010

0.837 0.629 0.003 0.002 0.005 0.008 0.002 0.004 0.006 0.007 0.003 0.009 0.006 0.001 0.003 0.006 0.002 0.003

0.916 1.122 0.003 0.002 0.004 0.005 0.002 0.001 0.004 0.009 0.002 0.006 0.001 0.006 0.003 0.005 0.001 0.001

0.005 0.007 0.001 0.001 0.004 0.002 0.001 0.003 0.002 0.005 0.009 0.002 0.001 0.004 0.004 0.002 0.009 0.007

Activitya on:

Strain

PAO1 PAO1(pRO103) PAO1(pRO1945) PAO1(pRO103, pRO1945) PAO1(pRO1944) PAO1(pRO103, pRO1944)

C?

TFD-

TFD-

CAA

PYR

CAA

y Tyr

0.001 0.004 0.520 0.567 0.762 0.776

0.001 0.001 0.003 0.503 0.701 0.791

0.006 0.001 0.504 0.583 0.523 0.525

0.001 0.006 1.067 0.648 0.884 1.062

Y

Strain

PYR

0.001 0.004

0.000 0.008 0.768 0.772

a Values are units of enzyme activity (as defined in the text) per milligram of protein. Each value is the mean of three independent determinations.

Tyr induction of MAA reductase activity. The results described above indicated that Tyr or a downstream metabolite of Tyr induces MAA reductase activity when the cloned mar gene from Pseudomonas sp. strain PKO1 is carried in cells of P. aeruginosa or P. putida. To determine whether Tyr induces MAA reductase activity in other bacterial strains, we transferred pRO103 by conjugal mating to a variety of gram-negative pseudomonads and assayed the plasmid-bearing transconjugants and the non-plasmidbearing strains for MAA reductase activity in cells grown on TFD-CAA, TFD-PYR, CAA, Tyr, or PYR. MAA reductase activity was present in all TFD-grown strains carrying pRO103, regardless of whether the ancillary carbon source was CAA or PYR, with the exceptions of P. aeruginosa and the two strains of P. putida (Table 4). However, when cells were grown on CAA or Tyr, MAA reductase activity was detected only in Pseudomonas sp. strain PKO1. None of the strains had significant levels of MAA reductase activity when grown on PYR. We also observed that MAA reductase activity was induced in CAA- or Tyr-grown cells of Pseudomonas sp. strain PKO1 that did not carry plasmid pRO103 (Table 4). This was not true for cells of A. eutrophus AEO106, the three strains of P. cepacia (DBO1, TL249, and PC383), or P. pickettii PKO2, which had shown inducible levels of MAA reductase activity when carrying plasmid pRO103 and grown on TFD. These results suggest that MAA reductase functions in the catabolism of Tyr in Pseudomonas sp. strain PKO1 and that the Tyr catabolic pathway in this strain differs from that found in the other pseudomonads used in this study. Chloride release. As expected,, chloride was released in stoichiometric amounts from 4CC by crude extracts of TFD-grown P. putida PP0300 cells carrying either pRO103 or pRO1946, which contains the TFD catabolic genes from

PKO1 PKO1(pRO103) AEO106 AEO106(pRO103) DBO1 DBO1(pRO103) TL249 TL249(pRO103) PC383 PC383(pRO103) PKO2 PKO2(pRO103) PAO1 PAO1(pRO103) PP0200 PP0200(pRO103) PP0300

PP0300(pRO103)

TFD-

TFD-

CAA

1.057 1.187 0.001 0.700 0.006 0.556 0.010 0.557 0.002 0.934 0.005 0.951 0.001 0.004 0.001 0.005 0.004 0.003

a See Table 2, footnote a.

the BamHI C fragment of pRO103 (Table 5). Chloride was also released stoichiometrically from 4CC by extracts of TFD-grown cells of P. putida PPO300 carrying either pRO103 or pRO1946 and also the mar-containing plasmid pRO1944 or pRO1945 in trans. In contrast, only 1 mol of chloride was released per mol of 45DCC added to TFD-grown cells of P. putida carrying either pRO103 or pRO1946 alone. However, when the mar-containing plasmid pRO1944 or pRO1945 was present in trans together with pRO103 or pRO1946, 2 mol of chloride was released per mol of 45DCC added (Table 5). DISCUSSION Chapman and Ribbons (5) showed that MAA reductase is involved in resorcinol catabolism by a strain of P. putida; however, Pseudomonas sp. strain PKO1 does not grow on resorcinol, nor do A. eutrophus AEO106, P. cepacia DBO1, TL249, or PC383, or P. pickettii PKO2. The results obtained TABLE 5. Chloride released from 4CC and 45DCC by cell extracts from P. putida PP0300 grown on TFD and carrying pRO103, the BamHI C fragment of pRO103, or the cloned mar gene from Pseudomonas sp. strain PKO1

Asustaye

Plasmid(s) Plasmid(s) substrate TABLE 3. Activity of MAA reductase cloned from Pseudomonas sp. strain PKO1 in P. putida PP0300

Activity" on: Strain

PP0300 PPO300(pRO103) PPO300(pRO1945) PP0300(pRO103, pRO1945) PP0300(pRO1944) PPO300(pRO103, pRO1944) * See Table 2, footnote a.

DCAA

PYR

0.004 0.001 0.003 0.002 0.935 0.005 0.393 0.302 1.065 1.032 1.126 1.013

CAA

Tyr

PYR

0.002 0.001

0.009

0.003 0.997 0.459 1.080 0.803

0.007 0.009 0.001 0.977 0.875

0.001 1.952 0.334 1.138 1.100

pRO103 pRO103 pRO1946 pRO1946 pRO103 + pRO1945 pRO103 + pRO1945 pRO103 + pRO1944 pRO103 + pRO1944 pRO1946 + pRO1945 pRO1946 + pRO1945 pRO1946 + pROJ944 pRO1946 + pRO1944

mol of Clof substrate addeda released/mola

4CC 45DCC 4CC 45DCC 4CC 45DCC 4CC

45DCC 4CC 45DCC 4CC 45DCC

" Each value is the mean of three independent determinations.

1.0 1.0 0.9 0.9 0.9 1.9 1.1 2.1 1.0 2.1 1.1 1.9

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in the present study suggest that MAA reductase activity is induced in Pseudomonas sp. strain PKO1 in the presence of Tyr or a degradation product of Tyr and that the Tyr catabolic pathway is the source from which MAA reductase is recruited for the degradation of the CMA produced from TFD in pRO103-bearing cells of Pseudomonas sp. strain PKO1. It should also be noted (Table 4) that pRO103-bearing cells of A. eutrophus AEO106, the three strains of P. cepacia (DBO1, TL249, and PC383), and P. pickettii PKO2 contain MAA reductase activity when grown on TFD but not when grown on Tyr. These results demonstrate that MAA reductase activity, which is required for the complete degradation of TFD, is recruited from a different source in these strains than in Pseudomonas sp. strain PKO1. Furthermore, these results indicate either that there is a difference in the pathways for Tyr catabolism between Pseudomonas sp. strain PKO1 and the other pseudomonads used in this study or that the CMA reductase activity induced in permissive strains is not derived from a Tyr degradation pathway. In the catabolic sequence proposed for the degradation of TFD by plasmid pJP4, TFD is converted to CMA by plasmid-encoded enzymes (10, 29). Two catabolic routes have been proposed for the further metabolism of CMA. One involves an NADH-dependent reduction to form 5-chloro3-oxoadipate, which is then cleaved to chlorosuccinate and acetyl coenzyme A (11). The second route (21) involves the reduction of CMA to ,-ketoadipate with the consumption of 2 mol of NADH and the liberation of 1 mol of chloride. Acetyl coenzyme A and succinate are then formed from .-ketoadipate via the conventional P-ketoadipate pathway. Since CMA was not available as a substrate, we could not directly determine which pathway was used by pRO103bearing cells of Pseudomonas sp. strain PKO1 grown on TFD. Our studies were conducted with MAA as a substrate, but apparently the cloned MAA reductase from Pseudomonas sp. strain PKO1 reduces CMA as well, as suggested by the induction of MAA reductase activity in TFD-PYR-grown cells. This conclusion was supported by the observation that when the mar gene cloned from Pseudomonas sp. strain PKO1 was present in pRO103-bearing cells of P. putida PP0300, these cells released stoichiometrically 2 mol of chloride per mol of 45DCC provided. Similarly, stoichiometric amounts of chloride were released when cells carried either plasmid pRO1944 or plasmid pRO1945 together with the BamHI C fragment of pRO103, which contains the gene cluster encoding enzymes for the conversion of 45DCC to CMA. These results suggest that the cloned MAA reductase from Pseudomonas sp. strain PKO1 can dehalogenate CMA. The foregoing results point to an interesting aspect of the enzymology of dehalogenation of chlorinated MAA by MAA reductase. The degradation of TFD by plasmid pRO103 results in the production of CMA as an intermediate, whereas the degradation of 45DCC results in the production of 3-chloromaleylacetate. Both of these chlorinated MAAs are utilized by MAA reductase, as shown by the results in Tables 3 and 5, suggesting that dechlorination occurs as a result of the reduction of MAA to ,B-ketoadipate. The two DNA fragments cloned from the chromosomal DNA of Pseudomonas sp. strain PKO1 could represent separate and independent fragments of the chromosome carrying genes for MAA reductase. Alternatively, the 0.5-kb DNA fragment, cloned as plasmid pRO1944, could represent an internal deletion of the larger BamHI fragment, cloned as plasmid pRO1945, which occurred during the cloning procedure. Restriction endonuclease digestion of the BamHI-SalI terminus (map coordinates 5.5 to 6.0 kb [Fig. 1]) of

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pRO1955, which was derived by deletion subcloning from pRO1945, indicated a distribution of restriction sites identical to that obtained in pRO1944 (Fig. 1). Therefore, we conclude that the two cloned DNA fragments are identical but that the pRO1944 fragment represents an internal deletion of the pRO1945 fragment. Such internal deletions are commonly observed with our pRO1727-based Pseudomonas cloning system (R. H. Olsen, unpublished data). Based on the foregoing suggestion that plasmids pRO1944 and pRO1945 are identical clones, our observation that MAA reductase activity is expressed constitutively in cells of P. aeruginosa or P. putida carrying plasmid pRO1944 suggests that the expression of MAA reductase is negatively controlled. This conclusion is further supported by the findings that cells of P. aeruginosa or P. putida carrying plasmid pRO1945 show no induction of MAA reductase activity when grown on PYR but do show a significant induction of MAA reductase activity when grown on TFD and that plasmid pRO1955, which contains a 1.0-kb Sall subclone of plasmid pRO1945, also exhibits MAA reductase activity constitutively. These results indicate that a regulatory locus that is present on pRO1945 and that controls the transcription of the mar gene is absent from plasmids pRO1944 and pRO1955. Furthermore, MAA reductase is induced by the CMA produced from TFD degradation and also by Tyr or a metabolite of Tyr. Extracts of CAA-grown cells of P. aeruginosa carrying plasmid pRO1944 or pRO1945 exhibited in SDS-polyacrylamide gels a novel polypeptide with an estimated molecular weight of 18,000. The amount of DNA required for the synthesis of this polypeptide corresponds to the size of the chromosomal BamHI fragment in plasmid pRO1944 as well as the corresponding Sall deletion subclone derived from plasmid pRO1945 (Fig. 1). From these data, we conclude that the novel 18,000-molecular-weight polypeptide produced by cells carrying plasmid pRO1944 or pRO1945 is the MAA reductase of Pseudomonas sp. strain PKO1. MAA reductase has been found to be an intermediate in the catabolism of Tyr by the yeast Trichosporon cutaneum (31). The enzyme has also been purified and characterized from resorcinol-grown cells of this yeast (15); however, the yeast enzyme differs significantly in molecular weight from the one that we have isolated. Recently, Weisshaar et al. (34) reported the cloning of DNA fragments containing genes for the degradation of chlorocatechol through ,-ketoadipate from a 3-chlorobenzoate-utilizing strain, Pseudomonas sp. strain B13 WRl. Although MAA reductase would be involved in the degradation of 3-chlorobenzoate, it is not clear from their work whether MAA reductase was encoded by the cloned DNA fragments or whether it was encoded by, and recruited from, the host cell in which the cloned DNA fragments were expressed. ACKNOWLEDGMENTS This work was supported in part by grants from the Michigan Biotechnology Institute, the University of Michigan Office of the Vice President for Research, and U.S. Environmental Protection Agency Cooperative Agreement CR-812679. We thank Peter Chapman for helpful discussions during the course of this investigation. LITERATURE CITED 1. Amy, P. S., J. W. Schulke, L. M. Frazier, and R. J. Seidler. 1985. Characterization of aquatic bacteria and cloning of genes

specifying partial degradation of 2,4-dichlorophenoxyacetic acid. Appl. Environ. Microbiol. 49:1237-1245.

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