Showing papers on "Pseudomonas putida published in 1988"

Degradation of trichloroethylene by toluene dioxygenase in whole-cell studies with Pseudomonas putida F1.

Abstract: Toluene-induced cells of Pseudomonas putida F1 removed trichloroethylene from growth media at a significantly greater initial rate than the methanotroph Methylosinus trichosporium OB3b. With toluene-induced P. putida F1, the initial degradation rate varied linearly with trichloroethylene concentration over the range of 8 to 80 microM (1.05 to 10.5 ppm). At 80 microM (10.5 ppm) trichloroethylene and 30 degrees C, the initial rate was 1.8 nmol/min per mg of total cell protein, but the rate decreased rapidly with time. A series of mutant strains derived from P. putida F1 that are defective in the todC gene, which encodes the oxygenase component of toluene dioxygenase, failed to degrade trichloroethylene and to oxidize indole to indigo. A spontaneous revertant selected from a todC culture regained simultaneously the abilities to oxidize toluene, to form indigo, and to degrade trichloroethylene. The three isomeric dichloroethylenes were degraded by P. putida F1, but tetrachloroethylene, vinyl chloride, and ethylene were not removed from incubation mixtures.

Survival of rifampin-resistant mutants of Pseudomonas fluorescens and Pseudomonas putida in soil systems

Abstract: The fate of spontaneous chromosomal rifampin-resistant (Rifr) mutants of Pseudomonas putida and Pseudomonas fluorescens in sterile and live organic soil from which they were isolated was studied. In sterile native-soil assays, a Rifr mutant of P. putida showed no decrease in competitive fitness when compared with the wild-type parent. However, mutants of P. fluorescens were of two general categories. Group 1 showed no difference from the wild type in terms of growth rate, competitive fitness, and membrane protein composition. Group 2 showed a slower growth rate in both minimal and enriched media and an altered membrane protein profile. These mutants also demonstrated decreased competitive fitness compared with the wild-type strain. In live soil, the Rifr P. putida strain persisted throughout the 38-day test period with a decay rate of 0.7 log10 CFU/g of soil per 10 days. A group 1 Rifr P. fluorescens mutant maintained its inoculated titer for 7 to 10 days and then decayed at a rate of 0.2 to 0.4 log10 CFU/g of soil per 10 days. A group 2 Rifr P. fluorescens mutant remained at its titer for 1 to 5 days before decaying at a two- to threefold-faster rate. These findings indicate that rifampin resistance may not be an innocuous mutation in some pseudomonads and that marked strains should be compared with wild-type parents before being used as monitors of parental strain survival. Colonization of sterile soil with either the wild-type or mutant strain precluded normal colonization of the second added strain.(ABSTRACT TRUNCATED AT 250 WORDS) Images

Toluene degradation by Pseudomonas putida F1: genetic organization of the tod operon.

Abstract: Pseudomonas putida PpF1 degrades toluene through cis-toluene dihydrodiol to 3-methylcatechol. The latter compound is metabolized through the well-established meta pathway for catechol degradation. The first four steps in the pathway involve the sequential action of toluene dioxygenase (todABC1C2), cis-toluene dihydrodiol dehydrogenase (todD), 3-methylcatechol 2,3-dioxygenase (todE), and 2-hydroxy-6-oxo-2,4-heptadienoate hydrolase (todF). The genes for these enzymes form part of the tod operon which is responsible for the degradation of toluene by this organism. A combination of transposon mutagenesis of the PpF1 chromosome, as well as analysis of cloned chromosomal fragments, was used to determine the physical order of the genes in the tod operon. The genes were determined to be transcribed in the order todF, todC1, todC2, todB, todA, todD, todE.

Trichloroethylene metabolism by microorganisms that degrade aromatic compounds

Abstract: Trichloroethylene (TCE) was metabolized by the natural microflora of three different environmental water samples when stimulated by the addition of either toluene or phenol. Two different strains of Pseudomonas putida that degrade toluene by a pathway containing a toluene dioxygenase also metabolized TCE. A mutant of one of these strains lacking an active toluene dioxygenase could not degrade TCE, but spontaneous revertants for toluene degradation also regained TCE-degradative ability. The results implicate toluene dioxygenase in TCE metabolism.

Specificity of pyoverdine-mediated iron uptake among fluorescent Pseudomonas strains.

Abstract: Pyoverdine-mediated iron transport was determined for seven fluorescent Pseudomonas strains belonging to different species. For all strains, cell or cell outer membrane and iron(III)-pyoverdine combinations were compared with their homologous counterparts in uptake, binding, and cross-feeding experiments. For four strains (Pseudomonas putida ATCC 12633, Pseudomonas fluorescens W, P. fluorescens ATCC 17400, and Pseudomonas tolaasii NCPPB 2192), the pyoverdine-mediated iron transport appeared to be strictly strain specific; pyoverdine-facilitated iron uptake by iron-starved cells and binding of ferripyoverdine to the purified outer membranes of such cells were efficient only in the case of the homologous systems. Cross-feeding assays, in liquid or solid cultures, resulted, however, especially for P. fluorescens ATCC 17400, in some discrepancies compared with uptake and binding assays, suggesting that growth experiments are the least likely to yield correct information on specificity of the pyoverdine-mediated iron transport. For the three other strains (P. fluorescens ATCC 13525, P. chlororaphis ATCC 9446, and P. aeruginosa ATCC 15692), cross-reactivity was demonstrated by the uptake, binding, and cross-feeding experiments. In an attempt to determine which parts of the iron transport system were responsible for the specificity, the differences in amino acid composition of the pyoverdines, together with the differences observed at the level of the iron-sensitive outer membrane protein pattern of the seven strains, are discussed.

Enantioselective synthesis through microbial oxidation of arenes. 1. Efficient preparation of terpene and prostanoid synthons

Abstract: Le cis-toluenediol est obtenu par biodegradation du toluene par Pseudomonas putida, souche 39D, et est un substrat pour la synthese de prostaglandine

Transfer and occurrence of large mercury resistance plasmids in river epilithon. [Pseudomonas fluorescens; Pseudomonas putida]

Abstract: In situ mating experiments were done in the River Taff, South Wales, United Kingdom, by using a natural mercury resistance plasmid (pQM1) isolated from a mixture of epilithic bacteria in vitro. The river temperature from March to November was found to influence transfer frequencies strongly (6.8 x 10/sup -9/ to 1.5 x 10/sup -2/ per recipient). A linear relationship existed between log/sub 10/ transfer frequency and river temperature (6 to 21/sup 0/C), a 2.6/sup 0/C change in temperature giving a 10-fold change in transfer frequency. In vitro experiments showed that pQM1 transferred most efficiently between fluorescent pseudomonads and that one epilithic isolate (Pseudomonas fluorescens) was an efficient donor in situ. Experiments with a P. putida recipient showed that intact epilithic bacterial communities could transfer mercury resistance plasmids in situ at frequencies of up to 3.75 x 10/sup -6/ per recipient. Nineteen of the large (>250-kilobase) plasmids isolated by transfer into P. putida were studied in detail and grouped into seven types by restriction digests. Mercury resistance and UV resistance were found to be common linked phenotypes in 19 of the 23 plasmids tested.

Oxidation of substituted phenols by Pseudomonas putida F1 and Pseudomonas sp. strain JS6

Abstract: The biodegradation of benzene, toluene, and chlorobenzenes by Pseudomonas putida involves the initial conversion of the parent molecules to cis-dihydrodiols by dioxygenase enzyme systems. The cis-dihydrodiols are then converted to the corresponding catechols by dihydrodiol dehydrogenase enzymes. Pseudomonas sp. strain JS6 uses a similar system for growth on toluene or dichlorobenzenes. We tested the wild-type organisms and a series of mutants for their ability to transform substituted phenols after induction with toluene. When grown on toluene, both wild-type organisms converted methyl-, chloro-, and nitro-substituted phenols to the corresponding catechols. Mutant strains deficient in dihydrodiol dehydrogenase or catechol oxygenase activities also transformed the phenols. Oxidation of phenols was closely correlated with the induction and activity of the toluene dioxygenase enzyme system.

Trichloroethylene metabolism by microorganisms that degrade aromatic compounds. [Pseudomonas putida]

Abstract: Trichloroethylene (TCE) was metabolized by the natural microflora of three different environmental water samples when stimulated by the addition of either toluene or phenol. Two different strains of Pseudomonas putida that degrade toluene by a pathway containing a toluene dioxygenase also metabolized TCE. A mutant of one of these strains lacking an active toluene dioxygenase could not degrade TCE, but spontaneous revertants for toluene degradation also regained TCE-degradative ability. The results implicate toluene dioxygenase in TCE metabolism.

Enumeration of Tn5 mutant bacteria in soil by using a most-probable-number-DNA hybridization procedure and antibiotic resistance

Abstract: Investigations were made into the utility of DNA hybridization in conjunction with a microdilution most-probable-number procedure for the enumeration of Rhizobium spp. and Pseudomonas putida in soil. Isolates of Rhizobium spp. and P. putida carrying the transposon Tn5 were added to sterile and nonsterile Burbank sandy loam soil and enumerated over time. Soil populations of rhizobia were enumerated by colony hybridization, most-probable-number-DNA hybridization procedure, plate counts, plant infectivity most probable number, and fluorescent antibody counts. Population values compared well for all methods at 5 and 30 days after the addition of cells, although the fluorescent antibody method tended to overestimate the viable population. In nonsterile soil, most-probable-number-DNA hybridization procedure enumerated as few as 10 P. putida Tn5 cells g of soil-1 and 100 R. leguminosarum bv. phaseoli Tn5 cells g of soil-1 and should have utility for following the fate of genetically engineered microorganisms released to the environment. Among the Kmr isolates containing Tn5, approximately 5% gave a dark, more intense autoradiograph when probed with 32P-labeled pGS9 DNA, which facilitated their detection in soil. Hybridization with a pCU101 probe (pGS9 without Tn5) indicated that donor plasmid sequences were being maintained in the bacterial chromosome. Transposon-associated antibiotic resistance was also utilized as a phenotypic marker. Tn5 vector-integrate mutants were successfully enumerated at low populations (10 to 100 cells g of soil-1) in soil by both phenotypic (Kmr) and genotypic (DNA probe) analysis. However, determination of the stability of Tn5 or Tn5 and vector sequences in the bacteria is necessary.

Molecular Studies on the Role of a Root Surface Agglutinin in Adherence and Colonization by Pseudomonas putida.

Abstract: Pseudomonas putida aggressively colonizes root surfaces and is agglutinated by a root surface glycoprotein. Mutants of P. putida derived chemically or by Tn5 insertion demonstrated enhanced or decreased agglutinability. Two nonagglutinable Tn5 mutants (Agg−) and two mutants with enhanced agglutinability (Aggs) possessed Tn5 in unique restriction sites. Agg− mutants colonized root surfaces of seedlings grown from inoculated seeds, but at levels lower than those observed with the Agg+ parent. In short-term binding studies, Agg− cells adhered at levels that were 20- to 30-fold less than those for Agg+ parental cells. These data suggest that the agglutination interaction plays a role in the attachment of P. putida to root surfaces.

Degradation of 1,4-dichlorobenzene by enriched and constructed bacteria

Abstract: Three strains, RHO1, R3 and B1, tentatively identified as a Pseudomonas sp., an Alcaligenes sp. and a Pseudomonas sp. which were able to use 1,4-dichlorobenzene as the sole carbon and energy source were isolated from water of the Rhine river and from the sewage plant at Leverkusen-Burrig. A hybrid strain, WR1313, which uses chlorobenzene as the growth substrate, was obtained by mating the benzene-growing Pseudomonas putida strain F1 with strain B13, a Pseudomonas sp. degrading chlorocatechols. Further selection of this strain for growth on 1,4-dichlorobenzene allowed the isolation of strain WR1323. During growth on 1,4-dichlorobenzene the strains released stoichiometric amounts of chloride. The affinity of the organisms to 1,4-dichlorobenzene was measured with strain R3 showing a Ks value of 1.2 mg/l. Respiration data and enzyme activities in cell extracts as well as the isolation of 3,6-dichlorocatechol from the culture fluid are consistent with the degradation of 1,4-dichlorobenzene via 3,6-dichlorocatechol, 2,5-dichloro-cis,cis-muconate, 2-chloro-4-carboxymethylenebut-2-en-4-olide.

Cloning and expression of pca genes from Pseudomonas putida in Escherichia coli.

Abstract: Summary: β-Ketoadipate elicits expression of five structural pca genes encoding enzymes that catalyse consecutive reactions in the utilization of protocatechuate by Pseudomonas putida. Three derivatives of P. putida PRS2000 were obtained, each carrying a single copy of Tn5 DNA inserted into a separate region of the genome and preventing expression of different sets of pca genes. Selection of Tn5 in or near the pca genes in these derivatives was used to clone four structural pca genes and to enable their expression as inserts in pUC19 carried in Escherichia coli. Three of the genes were clustered as components of an apparent operon in the order pcaBDC. This observation indicates that rearrangement of the closely linked genes accompanied divergence of their evolutionary homologues, which are known to appear in the order pcaDBC in the Acinetobacter calcoaceticus pcaEFDBCA gene cluster. Additional evidence for genetic reorganization during evolutionary divergence emerged from the demonstration that the P. putida pcaE gene lies more than 15 kilobase pairs (kbp) away from the pcaBDC operon. An additional P. putida gene, pcaR, was shown to be required for expression of the pca structural genes in response to β-ketoadipate. The regulatory pcaR gene is located about 15 kbp upstream from the pcaBDC operon.

Siderophore-mediated uptake of Fe3+ by the plant growth-stimulating Pseudomonas putida strain WCS358 and by other rhizosphere microorganisms.

Abstract: Under iron-limited conditions, Pseudomonas putida WCS358 produces a siderophore, pseudobactin 358, which is essential for the plant growth-stimulating ability of this strain. Cells of strain WCS358, provided that they have been grown under Fe3+ limitation, take up 55Fe3+ from the 55Fe3+-labeled pseudobactin 358 complex with Km and Vmax values of 0.23 microM and 0.14 nmol/mg of cell dry weight per min, respectively. Uptake experiments with cells treated with various metabolic inhibitors showed that this Fe3+ uptake process was dependent on the proton motive force. Furthermore, strain WCS358 was shown to be able to take up Fe3+ complexed to the siderophore of another plant-beneficial P. fluorescens strain, WCS374. The tested pathogenic rhizobacteria and rhizofungi were neither able to grow on Fe3+-deficient medium in the presence of pseudobactin 358 nor able to take up 55Fe3+ from 55Fe3+-pseudobactin 358. The same applies for three cyanide-producing Pseudomonas strains which are supposed to be representatives of the minor pathogens. These results indicate that the extraordinary ability of strain WCS358 to compete efficiently for Fe3+ is based on the fact that the pathogenic and deleterious rhizosphere microorganisms, in contrast to strain WCS358 itself, are not able to take up Fe3+ from Fe3+-pseudobactin 358 complexes.

Kinetics of the simultaneous batch degradation of p -cresol and phenol by Pseudomonas putida

Abstract: The kinetics of the batch degradation of phenol and p-cresol in a dual-substrate system by Pseudomonas putida was investigated. A model was developed which was able to successfully predict phenol and p-cresol levels during batch growth. A constant specific growth rate (μ) was assumed together with the assumption that the biomass yield factors (Y) for growth on phenol and p-cresol were identical. These simplifying assumptions allowed an analytical solution to the model equations to be easily obtained, as well as reducing the number of unknown parameters that required estimation. Phenol and p-cresol were metabolized simultaneously by P. putida. P. putida did not appear to metabolize either of the two substrates preferentially. The rate of utilization of aparticular organic substrate was related to its fraction of the total organic substrate present, i.e., for substrate 1 the assimilation rate depended on S 1/(S 1+S 2), where S iis the concentration of the ith substrate.

Transfer and expression of mesophilic plasmid-mediated degradative capacity in a psychrotrophic bacterium

Abstract: A psychrotrophic bacterium, originally isolated from a natural aquatic environment, was characterized and identified as Pseudomonas putida Q5 for use as a representative recipient for biodegradative genes from a mesophilic microorganism. The TOL plasmid pWWO of the mesophile P. putida PaW1 was successfully transferred by conjugation to the naturally isolated psychrotroph P. putida Q5, as shown by plasmid analysis by agarose gel electrophoresis. Expression of the genes encoded by the mesophilic TOL plasmid in the psychrotroph was shown by the fact that the transconjugant (designated P. putida Q5T) had the capacity to degrade and utilize toluate (1,000 mg/liter) as a sole source of carbon at temperatures as low as 0 degrees C. Comparison of growth rates over a wide temperature range (0 to 30 degrees C) indicated that the physiological activity of the transconjugant was not reduced and that the plasmid DNA from the mesophile and its encoded enzymes functioned effectively in the psychrotroph at temperatures well below those at which the mesophile could grow. The production and demonstrated functioning of P. putida Q5T illustrates the possibility of developing specific degradative capacities in bacteria which can readily function at low temperatures in chemically contaminated environments or in industrial wastewater treatment systems.

Transcriptional regulation, nucleotide sequence, and localization of the promoter of the catBC operon in Pseudomonas putida.

Abstract: The catB and catC genes encode cis,cis-muconate lactonizing enzyme I (EC 5.5.1.1) and muconolactone isomerase (EC 5.3.3.4), respectively. These enzymes are required for the dissimilation of benzoate to beta-ketoadipate by Pseudomonas putida and are under coordinate transcriptional regulation. By deletion analysis and the use of pKT240 as a promoter probe vector, we located a single promoter region for the catBC operon upstream of catB. RNA-DNA hybridization studies, together with reverse transcriptase mapping, demonstrated that this promoter must be activated in the presence of an inducer molecule for effective transcription of the operon. In addition, the transcription initiation site was located 64 base pairs upstream of the catB initiation codon, and sequences upstream of -43 were required for promoter function. The catBC promoter was compared with other positively regulated procaryotic promoters to identify possible consensus sequences.

Bacterial metabolism of side chain fluorinated aromatics: cometabolism of 3-trifluoromethyl(TFM)-benzoate by Pseudomonas putida (arvilla) mt-2 and Rhodococcus rubropertinctus N657

Abstract: The TOL plasmid-encoded enzymes of the methyl-benzoate pathway in Pseudomonas putida mt-2 cometabolized 3-trifluoromethyl (TFM)-benzoate. Two products, 3-TFM-1,2-dihydroxy-2-hydrobenzoate (3-TFM-DHB) and 2-hydroxy-6-oxo-7,7,7-trifluoro-hepta-2,4-dienoate (7-TFHOD) were identified chemically and by spectroscopic properties. TFM-substituted analogues of the metabolites of the methylbenzoate pathway were generally converted at drastically reduced rates. The catechol-2,3-dioxygenase from Pseudomonas putida showed moderate turnover rates with 3-TFM-catechol. The catechol-1,2-dioxygenase of Rhodococcus rubropertinctus N657 was totally inhibited by 3-TFM-catechol and did not cleave this substrate. Hammett-type analysis showed the catechol-1,2-dioxygenase reaction to be strongly dependent on the electronic nature of the substituents. Electronegative substituents strongly inhibited catechol cleavage. The catechol-2,3-dioxygenase reaction, however, was only moderately sensitive to electronegative substituents.

Loss of the Toluene-Xylene Catabolic Genes of TOL Plasmid pWWO during Growth of Pseudomonas putida on Benzoate Is Due to a Selective Growth Advantage of “Cured” Segregants

Abstract: Summary: During growth on benzoate-minimal medium Pseudomonas putida mt-2 (PaW1) segregates derivative (“cured”) strains which have lost the ability to use the pathway encoded by its resident catabolic plasmid pWWO. Experiments with two plasmids identical to pWWO but each with an insert of Tn401, which confers resistance to carbenicillin, suggested that the “benzoate caring” occurs far more frequently by the specific deletion of the 39 kbp region carrying the catabolic genes than by total plasmid loss. This effect was not pH-dependent, and was not produced during growth on other weak organic acids, such as succinate or propionate, or when benzoate was present in the medium with an alternative, preferentially used carbon source such as succinate. Growth on benzoate did not cause loss from strain PaW174 of the plasmid pWWO-174, a derivative of pWWO which has deleted the 39 kbp region but carries Tn401. Similarly the naphthalene-catabolic plasmid pWW60-1, of the same incompatibility group as pWWO, was not lost from PaW701 during growth on benzoate. Competition between wild-type PaW1 and PaW174, which has the “cured” phenotype, showed that the latter has a distinct growth advantage on benzoate over the wild-type even when initially present as only 1% of the population: when PaW174 was seeded at lower cell ratios, spontaneously “cured” derivatives of PaW1 took over the culture after 60-80 generations, indicating that they are present in PaW1 cultures at frequencies between 10−2 and 10−3. We conclude that the progressive takeover of populations of PaW1 only occurs when benzoate is present as the sole growth source and that neither benzoate, nor other weak acids, affect plasmid segregation or deletion events: a sufficient explanation is that the “cured” segregants grow faster than the wild-type using the chromosomally determined β-ketoadipate pathway.

Fusarium Wilt Suppression and Agglutinability of Pseudomonas putida.

Abstract: Mutants of Pseudomonas putida (Agg−) that lack the ability to agglutinate with components present in washes of bean and cucumber roots showed limited potential to protect cucumber plants against Fusarium oxysporum f. sp. cucumerinum. However, a higher level of protection was observed against Fusarium wilt in cucumber plants coinoculated with the parental bacterium (Agg+), which was agglutinable. The Agg− mutants did not colonize the roots of cucumber plants as extensively as the Agg+ parental isolate did. In competition experiments involving bean roots inoculated with a mixture of Agg+ and Agg− bacteria, the Agg+ strains colonized roots to a greater extent than the Agg− cells did. These data suggest that the Agg+ phenotype provides additional interactions that aid in the beneficial character of P. putida.

4-Methoxybenzoate monooxygenase from Pseudomonas putida: isolation, biochemical properties, substrate specificity, and reaction mechanisms of the enzyme components.

Abstract: Publisher Summary Studies on the mechanism of biological degradation of lignin or lignin model substances by fungi showed that degradation of lignin down to vanillic acid followed the pathway involving successively the intermediates: α-guaiacyl glycerolconiferyl ether, 4-hydroxy-3-methoxyphenylpyruvic acid, or 4-hydroxy-3-methoxycinnamic acid and vanillin. This chapter describes purification of the components of 4-methoxybenzoate monooxygenase. The enzyme activity of the cell-free crude extract is not proportional to protein concentration in the assay mixture, especially at low protein concentrations. This behavior shows that 4-methoxybenzoate monooxygenase is a dissociable enzyme system—that is, it consists of several components. This is confirmed by the isolation of two components, a reductase and a dioxygen-activating protein. The reconstitution of these two components reveals full enzymatic activity. The chapter also describes the properties of putidamonooxin (PMO). PMO, the dioxygen-activating component of the 4-methoxybenzoate monooxygenase, has a molecular weight of 126,000, as derived from ultracentrifugation and gel filtration.

Comparison of the amino acid sequences of the transacylase components of branched chain oxoacid dehydrogenase of Pseudomonas putida, and the pyruvate and 2‐oxoglutarate dehydrogenases of Escherichia coli

Abstract: The nucleotide sequence of bkdB, the structural gene for E2b, the transacylase component of branched-chain-oxoacid dehydrogenase of Pseudomonas putida has been determined and translated into its amino acid sequence. The start of bkdB was identified from the N-terminal sequence of E2b isolated from branched-chain-oxoacid dehydrogenase of the closely related species, P. aeruginosa. The reading frame was composed of 65.5% G + C with 82.3% of the codons ending in G or C. There was no intergenic space between bkdA2 and bkdB. No codons requiring minor tRNAs were utilized and the codon bias index indicated a preferential codon usage. The bkdB gene encoded 423 amino acids although the N-terminal methionine was absent from E2b prepared from P. aeruginosa. The relative molecular mass of the encoded protein was 45134 (45003 minus methionine) vs 47000 obtained by SDS/polyacrylamide gel electrophoresis. There was a single lipoyl domain in E2b compared to three lipoyl domains in E2p, and one domain in E2o, the transacylases of pyruvate and 2-oxoglutarate dehydrogenases of Escherichia coli respectively. There was significant similarity between the lipoyl domain of E2b and of E2p and E2o as well as between the E1-E2 binding domains of E2b, E2p and E2o. There was no similarity between the E3 binding domain of E2b to E2p and E2o which may reflect the uniqueness of the E3 component of branched-chain-oxoacid dehydrogenase of P. putida. The conclusions drawn from these comparisons are that the trans-acylases of prokaryotic pyruvate, 2-oxoglutarate and branched-chain-oxoacid dehydrogenases descended from a common ancestral protein probably at about the same time.

Comparison of the meta pathway operons on NAH plasmid pWW60-22 and TOL plasmid pWW53-4 and its evolutionary significance.

Abstract: Summary: The regulated meta pathway operon for the catabolism of salicylate on the naphthalene plasmid pWW60-22 was cloned into the broad-host-range vector pKT230 on a 17.5 kbp BamHI fragment. The recombinant plasmid conferred the ability to grow on salicylate when mobilized into plasmid-free Pseudomonas putida PaW130. A detailed restriction map of the insert was derived and the locations of some of the genes were determined by subcloning and assaying for their gene products in Escherichia coli and P. putida hosts. The existence of a regulatory gene was demonstrated by the induction of enzyme activities in the presence of salicylate. DNA-DNA hybridization indicated a high degree of structural homology between the pWW60-22 operon and the analogous meta pathway operon on TOL plasmid pWW53-4. The data are consistent with the structural genes being arranged in an identical linear array and suggest an evolutionary link between the two catabolic systems.