Degradative Plasmid Research Articles

Characterization of a new 2,4-dichlorophenoxyacetic acid degrading plasmid pEST4011: physical map and localization of catabolic genes

Plasmid pEST4011 enables Pseudomonas putida PaW85 to degrade 2,4-dichlorophenoxyacetic acid (2,4-D) and 3-chlorobenzoate (3-CBA). This new 2,4-D degradative plasmid has considerable homology with the regions of pJP4 containing the 2,4-D degradative genes (tfd). Restriction fragment BamHI-B of plasmid pEST4011, which has homology with this region, was cloned into the broad-host-range vector pKT240 and studied in P. putida PaW85. Restriction mapping, hybridization analysis and enzyme assays established the location of the genes for 2,4-D monooxygenase (tfdA), 2,4-dichlorophenol hydroxylase (tfdB), chlorocatechol 1,2-dioxygenase (tfdC) and the tfdR and tfdS regulatory genes on this fragment. Plasmid pEST4012 is a derivative of pEST4011 derived through the spontaneous deletion of a 42 kbp DNA fragment, which results in the loss of the 2,4-D+ and 3-CBA+ phenotype. We present here the physical maps of pEST4011 and pEST4012. In spite of the similarities in functions, the size (70 kbp), order of catabolic genes and restriction pattern of pEST4011 are clearly different from those of pJP4.

Regulation of the catechol 1,2-dioxygenase- and phenol monooxygenase-encoding pheBA operon in Pseudomonas putida PaW85

In Pseudomonas putida PaW85, the ortho-cleavage pathway is used for catechol degradation. The 11.4-kb XhoI fragment cloned from phenol degradation plasmid pEST1226 into pKT240 (recombinant plasmid pAT1140) contains the inducible pheBA operon that encodes catechol 1,2-dioxygenase (gene pheB) and phenol monooxygenase (gene pheA), the first two enzymes for the phenol degradation pathway. The promoter of the pheBA operon is mapped 1.5 kb upstream of the pheB gene. The plasmid pAT1140, when introduced into P. putida PaW85, enables the bacteria to use the hybrid plasmid-chromosome-encoded pathway for phenol degradation. The synthesis of the plasmid-encoded phenol monooxygenase and catechol 1,2-dioxygenase is induced by cis,cis-muconate. The expression studies of the deletion subclones derived from pAT1140 revealed that the transcription of the pheBA operon is positively controlled by a regulatory protein that is chromosomally encoded in P. putida. cis,cis-Muconate in cooperation with positive transcription factor CatR activates the transcription of the chromosomal ortho-pathway genes catA and catBC in P. putida (R. K. Rothmel, T. L. Aldrich, J. E. Houghton, W. M. Coco, L. N. Ornston, and A. M. Chakrabarty, J. Bacteriol. 172:922-931, 1990). The inability to express the pheBA operon in a P. putida CatR- background and activation of transcription of the pheBA operon in Escherichia coli in the presence of the catR-expressing plasmid demonstrated that the transcription of the pheBA operon in P. putida PaW85 carrying pEST1226 is controlled by the chromosomally encoded CatR.

Growth and plasmid-encoded naphthalene catabolism of Pseudomonas putida in batch culture.

The growth characteristics of Pseudomonas putida plasmid-harbouring strains which catabolize naphthalene via various pathways in batch culture with naphthalene as the sole source of carbon and energy have been investigated. The strains under study were constructed using the host strain P. putida BS394 which contained various naphthalene degradation plasmids. The highest specific growth rate was ensured by the plasmids that control naphthalene catabolism through the meta-pathway of catechol oxidation. The strains metabolizing catechol via the ortho-pathway grew at a lower rate. The lowest growth rate was observed with strain BS291 harbouring plasmid pBS4 which controls naphthalene catabolism via the gentistic acid pathway. Various pathways of naphthalene catabolism appear to allow these strains to grow at various rates which should be taken into account when constructing efficient degraders of polycyclic aromatic compounds.

Transposition of the TOL catabolic genes (Tn4651) into the degradative plasmid pSAH of Alcaligenes sp. O-1 ensures simultaneous mineralization of sulpho- and methyl-substituted aromatics

SUMMARY: Mixtures of 2-aminobenzenesulphonate (trivial name orthanilic acid, OA) and 3-methylbenzoic acid (3-MB), which are degraded by enzymes of plasmid-encoded pathways, can exert inhibition of growth and respiration in Alcaligenes sp. O-1 and Pseudomonas putida mt-2 depending on the ratio of their concentrations. The pronounced inhibition of Alcaligenes sp. O-1 growing on OA by the addition of equimolar amounts of 3-MB is characterized by a rapid inactivation of the OA-converting desulphonation activity. The exconjugant Alcaligenes sp. O/T was selected for simultaneous breakdown of OA and 3-MB by assembling the catabolic pathways from the plasmids pSAH (OA) and pWW0 (3-MB) of the above strains. The transpositional insertion of the TOL catabolic genes (Tn4651) from pWW0 into the recombinant plasmid of the exconjugant O/T was detected by Southern blot hybridization using the TOL plasmid as a probe. The exconjugant showed a rapid inactivation of OA desulphonation activity similar to the parent strain. However, following induction of the TOL catabolic genes and mineralization of 3-MB, the exconjugant O/T recovered and displayed high desulphonation activity, thus allowing sequential breakdown of both substrates. Our results clearly extend the expression range of the TOL catabolic genes, but not the replication ability of the plasmid, to the genus Alcaligenes.

Plasmids pJP4 and r68.45 Can Be Transferred between Populations of Bradyrhizobia in Nonsterile Soil

IncP plasmid r68.45, which carries several antibiotic resistance genes, and IncP plasmid pJP4, which contains genes for mercury resistance and 2,4-dichlorophenoxyacetic acid degradation, were evaluated for their ability to transfer to soil populations of rhizobia. Transfer of r68.45 was detected in nonsterile soil by using Bradyrhizobium japonicum USDA 123 as the plasmid donor and several Bradyrhizobium sp. strains as recipients. Plasmid transfer frequencies ranged up to 9.1 x 10 in soil amended with 0.1% soybean meal and were highest after 7 days with strain 3G4b4-RS as the recipient. Transconjugants were detected in 7 of 500 soybean nodules tested, but the absence of both parental strains in these nodules suggests that plasmid transfer had occurred in the soil, in the rhizosphere, or on the root surface. Transfer of degradative plasmid pJP4 was also evaluated in nonsterile soil by using B. japonicum USDA 438 as the plasmid donor and several Bradyrhizobium sp. strains as recipients. Plasmid pJP4 was transferred only when strains USDA 110-ARS and 3G4b4-RS were the recipients. The plasmid transfer frequency was highest for strain 3G4b4-RS (up to 7.4 x 10). Mercury additions to soil, ranging from 10 to 50 mug/g of soil, did not affect population levels of parental strains or the plasmid transfer frequency.

Polymerase chain reaction and gene probe detection of the 2,4-dichlorophenoxyacetic acid degradation plasmid, pJP4.

Specific and sensitive detection of indigenous and introduced degradative organisms is an essential prerequisite to their use in remediation of toxic waste and soil systems. Procedures were employed for the use of polymerase chain reaction and gene probes for sensitive detection of the 2,4-dichlorophenoxyacetic-acid-degrading bacterium, Alcaligenes eutrophus JMP134(pJP4). Two 20-mer oligonucleotide primers were identified for amplification of a 205-bp region of the tfdB gene of pJP4, and optimum conditions for amplification were determined. Both the polymerase chain reaction amplification process and hybridization with the 5'-end-labelled probe were found to be specific to organisms containing plasmid pJP4 or its derivative pRO103. Detection limits were determined for the template supplied either as bacterial cells or purified plasmid DNA. The detection was sensitive up to an initial inoculum of 3,000 CFU or 156 pg of total plasmid DNA. However, when the amplified product was transferred to a nylon membrane and hybridized with the 5'-end-labelled probe, the detection sensitivity increased to 300 CFU or 15.6 pg of plasmid DNA. This sensitive detection method is more specific than use of traditional indicator media (M. A. Loos, Can. J. Microbiol. 21:104-107, 1975). An oligonucleotide (20 bases) complementary to a sequence internal to the 205-bp region was synthesized and utilized as a probe to confirm the specificity of the detection.

Cross hybridization of plasmid and genomic DNA from aromatic and polycyclic aromatic hydrocarbon degrading bacteria

Forty-three bacterial strains were collected from various environmental and commercial sources and their ability to degrade polycyclic aromatic hydrocarbons (PAHs) was confirmed using the criteria of growth, mineralization, and oxidation. Undigested genomic DNA from these strains was blotted by Southern transfer to replicate membranes, which were probed either with purified plasmids (e.g., TOL and NAH7, associated with toluene and naphthalene degradation, respectively) or with genomic DNA from the other strains. The isolates were grouped according to hybridization and PAH-degradation results. One group of eight strains grew on naphthalene vapors as sole carbon source and hybridized with archetypical NAH plasmids. Another group of six isolates mineralized phenanthrene but could not grow on naphthalene, and their cryptic plasmids hybridized with Pseudomonas sp. HL7b, which degrades a wide range of PAHs. The remaining isolates, which could not grow on naphthalene but mineralized and (or) oxidized a variety of PAHs, hybridized with neither the pure plasmids nor heterologous genomic DNA, implying that their PAH-degradative genes were significantly dissimilar. This suggests that using TOL or NAH plasmids to probe an environmental population might reveal toluene- or naphthalene-degradative genes but would underestimate the occurrence of PAH-degradative genes. We suggest that a suite of probes would be necessary to evaluate the PAH-degradation potential of a mixed population. Key words: polycyclic aromatic hydrocarbons, degradative plasmids, NAH plasmid, TOL plasmid, hybridization.

Effect of 2-hydroxybenzoate on the maintenance of naphthalene-degrading pseudomonads in seeded and unseeded soil

The addition of specific nontoxic inducers of catabolic operons to contaminated sites is an approach that may enhance the efficiency of in situ biodegradation. We determined the genetic response of six pseudomonads to salicylate (also known as 2-hydroxybenzoate) added directly to 50 g of nonsterile soil samples. The strains, isolated from a polyaromatic hydrocarbon-contaminated soil, metabolized naphthalene as the sole source of available carbon, and their DNA sequences show significant homology to the nahAB genes of the degradative plasmid NAH7. Duplicate nonsterile soil cultures were incubated for up to 30 days. Experimental soil cultures were seeded with naphthalene-degrading strains (10(8) CFU g-1) originally isolated from the soil and amended with salicylate (16 or 160 micrograms g-1). Soil samples were analyzed periodically for the population density of heterotrophic bacteria and naphthalene degraders and for the abundance of the naphthalene-degradative genotype in the bacterial community. At 160 micrograms g-1, salicylate sustained the density of naphthalene degraders at the introduced density for 30 days in addition to producing a two- to sixfold increase in the occurrence in the bacterial community of DNA sequences homologous to the nah operon. No change in recoverable bacterial population densities was observed when soil samples were amended with 16 micrograms of salicylate g-1, but this concentration of salicylate induced a significant increase in the level of nah-related genes in the population.

Evidence of plasmid mediated dechlorinase activity in Pseudomonas sps.

The capability of strains of Pseudomonas ovalis (CFT1) and Pseudomonas tralucida (CFT4) to grow on hexachlorocyclohexane (HCH), a chlorinated pesticide, as sole source of carbon and energy is lost (though at a low frequency) by treatment with mitomycin C at a certain minimal inhibitory concentration (MIC). Presence of plasmid in HCH + culture and loss of degradative pathway and physical presence of plasmid upon curing indicate the extrachromosomal location of HCH degradative genes in CFT1. Furthermore, the HCH + degradation determinant of strains CM and CFT4 could be transferred to cured or to other HCH − Pseudomonads by conjugation without the transfer of any chromosomal marker of cooxidation of organochlorides. The metabolic activity, however, does not match the enormous quantity of the pesticides accumulated in nature over the years ( Rajchengappa and Rajghatta, 1989,). It is, therefore, imperative to identify such gene clusters and clone these, after appropriate manipulations, so that better recombinants are developed. Plasmids specifying biodegradation of a number of aromatics, including chlorinated compounds, are now known ( Rheinwald et al.1973 Fisher et al., 1978 Chatterjee et al., 1981). The degradative pathway for the organochlorides like 3 chlorobenzoate (3-CBA), 4-chlorobiphenyl, 2,4-dichlorophenoxyacetic acid (2,4-D) have been found to be controlled and expressed by an extrachromosomal gene cluster and plasmids bearing such clusters have been named ‘degradative plasmids’ ( Chatterjee et al., 1981 Furukawa and Chakrabarty, 1982 Pemberton and Fisher, 1977). Such degradative plasmids have been identified mainly in strains of Pseudomonas species which are already known for nutritional versatility ( Harayama and Don, 1979). The degradative plasmids are, by and large, conjugative types and their molecular sizes range from 40 to > 300 MDs ( Hardman et al., 1986). The large molecular size of these plasmids contributes to the difficulty associated with their isolation in covalently closed circular form. Furthermore, due to the low copy number of these plasmids, in situ identification of plasmid bands in agarose gel preparations is generally not possible. Special care is, therefore, necessary for obtaining consistent results with regard to identification of the degradative plasmids. Inter- and/or intra-generic conjugational transfer of these degradative plasmids have often been taken as a confirmation of the presence of such plasmids in a bacterial species. In the present communication two bacterial species of Pseudonionas, capable of complete degradation of HCH, have been studied for the presence of plasmids and their conjugative character.

Specific deletion of a large segment of pRA500: a 3,5-xylenol degradative plasmid

3,5-Xylenol degradative plasmid, pRA500 (approximately 500 kilobase pairs, kb) carried by Pseudomonas putida NCIB 9869, also encodes resistance to inorganic mercuric ions (Hgr). Following growth of Ps. putida on benzoate, p-cresol, p-hydroxybenzoate or protocatechuate, the frequency of loss of the 3,5-xylenol phenotype was 75–90%. The deletion of a large segment of approximately 350 kb from pRA500, to give rise to archetypal plasmid designated pRA502, occurred in such 3,5-xylenol-negative derivatives. During the conjugational transfer of pRA500 following selection of transconjugants for Hgronly, frequency of loss of 3,5-xylenol phenotype was approximately 60%. A high number of these 3,5-xylenol-negative transconjugants carried pRA502. A site-specific deletion is suggested in the formation of archetypal plasmid pRA502 by growth of Ps. putida on the above compounds and during transfer of pRA500.

In vivo interactions of the NahR transcriptional activator with its target sequences. Inducer-mediated changes resulting in transcription activation

The nahR gene from the NAH7 naphthalene degradation plasmid encodes a LysR-type transcriptional activator of the nah and sal promoters (Pnah and Psal, respectively) that responds to the inducer salicylate. In vivo methylation protection experiments with dimethyl sulfate showed that in the absence of inducer, NahR interacts in a similar manner with its target sites at Psal and Pnah. Both target sites also have very similar sequences comprised of a 4-base pair interrupted dyad containing two symmetrical guanines (-73 and -64 of Pnah; -71 and -62 of Psal), each located in adjacent major grooves on the same helical face, and both strongly protected by NahR. When inducer was present, several additional guanines of Pnah (-35, -45, and -58) and Psal (-42 and -40) became protected from methylation, while a guanine at -52 of Pnah became markedly enhanced for methylation, indicating that inducer and NahR-dependent interactions with these downstream sites of each promoter are quite different. Deletion of Psal sequences downstream of -30 did not affect its methylation patterns suggesting that NahR alone is responsible for the altered reactivities of these nucleotides. Similar in vivo methylation analyses with inducer-insensitive or inducer-independent NahR mutants also suggested that all alterations in methylation sensitivity are directly caused by NahR. It is more probable that the salicylate-induced reactivity changes result from direct NahR-guanine contacts which are required for, but not sufficient for transcription activation; however, they could also result from NahR-induced DNA contortions caused by upstream protein-DNA contacts.

Comparative genetic organization of incompatibility group P degradative plasmids

Plasmids that encode genes for the degradation of recalcitrant compounds are often examined only for characteristics of the degradative pathways and ignore regions that are necessary for plasmid replication, incompatibility, and conjugation. If these characteristics were known, then the mobility of the catabolic genes between species could be predicted and different catabolic pathways might be combined to alter substrate range. Two catabolic plasmids, pSS50 and pSS60, isolated from chlorobiphenyl-degrading strains and a 3-chlorobenzoate-degrading plasmid, pBR60, were compared with the previously described IncP group (Pseudomonas group P-1) plasmids pJP4 and R751. All three of the former plasmids were also members of the IncP group, although pBR60 is apparently more distantly related. DNA probes specific for known genetic loci were used to determine the order of homologous loci on the plasmids. In all of these plasmids the order is invariant, demonstrating the conservation of this "backbone" region. In addition, all five plasmids display at least some homology with the mercury resistance transposon, Tn501, which has been suggested to be characteristic of the beta subgroup of the IncP plasmids. Plasmids pSS50 and pSS60 have been mapped in detail, and repeat sequences that surround the suspected degradation genes are described.

Regulation of tfdCDEF by tfdR of the 2,4-dichlorophenoxyacetic acid degradation plasmid pJP4

The closely linked structural genes tfdCDEF borne on the 2,4-dichlorophenoxyacetic acid (TFD) catabolic plasmid, pRO101, were cloned into vector pRO2321 as a 12.6-kilobase-pair BamHI C fragment and designated pRO2334. The first gene in this cluster, tfdC, encodes chlorocatechol 1,2-dioxygenase and was expressed constitutively. Chlorocatechol 1,2-dioxygenase expression by pRO2334 was repressed in trans by the negative regulatory element, tfdR, on plasmid pRO1949. Derepression of tfdC was achieved when Pseudomonas aeruginosa PAO4032 containing both plasmids pRO2334 and pRO1949 was grown in minimal glucose medium containing TFD, 2,4-dichlorophenol, or 4-chlorocatechol, suggesting that TFD and other pathway intermediates can act as inducing compounds. Genetic organization of the tfdCDEF cluster was established by deletion of the tfdC gene, which resulted in the loss of tfdD and tfdE activity, suggesting that genes tfdCDEF are organized in an operon transcribed from the negatively regulated promoter of tfdC. Deletion subcloning of pRO1949 was used to localize tfdR to a 1.2-kilobase-pair BamHI-XhoI region of the BamHI E fragment of plasmid pRO101. The tfdR gene product was shown not to regulate the expression of tfdB, which encodes 2,4-dichlorophenol hydroxylase.

Use of saturation mutagenesis to localize probable functional domains in the NahR protein, a LysR-type transcription activator.

The NahR protein of the Pseudomonas naphthalene degradation plasmid NAH7 encodes a 300-residue transcription activator which is very similar to the NodD transcription activator of Rhizobium and other proteins in the LysR activator family. NahR binds to conserved sequences upstream (nucleotides -80 to -47) of the nah and sal promoters and activates transcription of genes for naphthalene catabolism in response to the inducer salicylate. Transformation of an Escherichia coli gal deletion strain (containing a sal promoter-galK fusion plasmid) with hydroxylamine-treated nahR DNA and selection on galactose/salicylate plates allowed isolation of 30 unique activation-deficient nahR alleles which fell into two classes: class I, defective in both activation and specific binding to the NahR activation site of the sal promoter; and class II, defective in activation, but with wild-type DNA binding activity. DNA sequence analysis showed that the amino acid substitutions eliminating DNA binding activity were mostly clustered in an NH2-terminal helix-turn-helix motif (residues 23-45) or a COOH-terminal domain (residues 239-291). Similar analysis of class II mutants identified a domain (residues 126-206) possibly involved in inducer binding and/or transcription activation functions. The partial trans-dominance of many mutant alleles and the size of NahR-specific DNA binding activity measured by gel filtration suggest that the active NahR protein may be a tetramer.

Location and organization of the dimethylphenol catabolic genes of Pseudomonas CF600

The gene organization of the phenol catabolic pathway of Pseudomonas CF600 has been investigated. This strain can grow on phenol and some methylated phenols by virtue of an inducible phenol hydroxylase and metacleavage pathway enzymes. The genes coding for these enzymes are located on pVI150, an IncP-2 degradative mega plasmid of this strain. Twenty-three kilobases of contiguous DNA were isolated from lambda libraries constructed from strains harbouring wild type and Tn5 insertion mutants of pVI150. A 19.9 kb region of this DNA has been identified which encodes all the catabolic genes of the pathway. Using transposon mutagenesis, polypeptide analysis and expression of subfragments of DNA, the genes encoding the first four enzymatic steps of the pathway have been individually mapped and found to lie adjacent to each other. The order of these genes is the same as that for isofunctional genes of TOL plasmid pWWO and plasmid NAH7.

Characterization of a phenanthrene degradation plasmid from Alcaligenes faecalis AFK2

A plasmid pHK2 involved in phenanthrene degradation was isolated from Alcaligenes faecalis AFK2. Some mitomycin C treated cells lost the pHK2 plasmid. The plasmid could be transformed into Pseudomonas putida and A. faecalis. They gained the ability to utilize phenanthrene as well as o-phthalate, an intermediate of phenanthrene degradation. Phenanthrene dioxygenase could be detected from the transformants after induction with phenanthrene. The molecular size of pHK2 DNA was determined to be 42.5 kilobase (kb), and its physical map was constructed.

Characterisation of a degradative plasmid in Pseudomonas putida that controls the expression of 2,4-xylenol degradative genes

Characterisation of a degradative plasmid in Pseudomonas putida that controls the expression of 2,4-xylenol degradative genes

Effect of degradative plasmid CAM-OCT on responses of Pseudomonas bacteria to UV light.

The effect of plasmid CAM-OCT on responses to UV irradiation was compared in Pseudomonas aeruginosa, in Pseudomonas putida, and in Pseudomonas putida mutants carrying mutations in UV response genes. CAM-OCT substantially increased both survival and mutagenesis in the two species. P. aeruginosa strains without CAM-OCT exhibited much higher UV sensitivity than did P. putida strains. UV-induced mutagenesis of plasmid-free P. putida was easily detected in three different assays (two reversion assays and one forward mutation assay), whereas UV mutagenesis of P. aeruginosa without CAM-OCT was seen only in the forward mutation assay. These results suggest major differences in DNA repair between the two species and highlight the presence of error-prone repair functions on CAM-OCT. A number of P. putida mutants carrying chromosomal mutations affecting either survival or mutagenesis after UV irradiation were isolated, and the effect of CAM-OCT on these mutants was determined. All mutations producing a UV-sensitive phenotype in P. putida were fully suppressed by the plasmid, whereas the plasmid had a more variable effect on mutagenesis mutations, suppressing some and producing no suppression of others. On the basis of the results reported here and results obtained by others with plasmids carrying UV response genes, it appears that CAM-OCT may differ either in regulation or in the number and functions of UV response genes encoded.

Nucleotide sequence and expression of clcD, a plasmid-borne dienelactone hydrolase gene from Pseudomonas sp. strain B13.

The clcD structural gene encodes dienelactone hydrolase (EC 3.1.1.45), an enzyme that catalyzes the conversion of dienelactones to maleylacetate. The gene is part of the clc gene cluster involved in the utilization of chlorocatechol and is carried on a 4.3-kilobase-pair BglII fragment subcloned from the Pseudomonas degradative plasmid pAC27. A 1.9-kilobase-pair PstI-EcoRI segment subcloned from the BglII fragment was shown to carry the clcD gene, which was expressed inducibly under the tac promoter at levels similar to those found in 3-chlorobenzoate-grown Pseudomonas cells carrying the plasmid pAC27. In this study, we present the complete nucleotide sequence of the clcD gene and the amino acid sequence of dienelactone hydrolase deduced from the DNA sequence. The NH2-terminal amino acid sequence encoded by the clcD gene from plasmid pAC27 corresponds to a 33-residue sequence established for dienelactone hydrolase encoded by the Pseudomonas sp. strain B13 plasmid pWR1. A possible relationship between the clcD gene and pcaD, a Pseudomonas putida chromosomal gene encoding enol-lactone hydrolase (EC 3.1.1.24) is suggested by the fact that the gene products contain an apparently conserved pentapeptide neighboring a cysteinyl side chain that presumably lies at or near the active sites; the cysteinyl residue occupies position 60 in the predicted amino acid sequence of dienelactone hydrolase.

Interposon mutagenesis of soil and water bacteria: a family of DNA fragments designed for in vitro insertional mutagenesis of Gram-negative bacteria

We have constructed a series of derivatives of the Ω interposon [Prentki and Krisch, Gene 29 (1984) 303–313 ] that can be used for in vitro insertional mutagenesis. Each of these DNA fragments carries a different antibiotic or Hg 2+ resistance gene (Ap R, Cm R, Tc R, Km R or Hg R) which is flanked, in inverted orientation, by transcription and translation termination signals and by synthetic polylinkers. The DNA of these interposons can be easily purified and then inserted, by in vitro ligation, into a plasmid linearized either at random by DNase I or at specific sites by restriction enzymes. Plasmid molecules which contain an interposon insertion can be identified by expression of its drug resistance. The position of the interposon can be precisely mapped by the restriction sites in the flanking polylinker. To verify their properties we have used these Ω derivatives to mutagenize a broad host range plasmid which contains the entire meta-cleavage pathway of the toluene degradation plasmid pWW0 of Pseudomonas putida. Insertion of these interposons in the plasmid between the promoter and the catechol 2,3-dioxygenase (C23O) gene dramatically reduced the expression of this enzyme in Escherichia coli. We also show that when a plasmid containing an ω interposon is transferred by conjugal mobilization from E. coli to P. putida, Agrobacterium tumefaciens, Erwinia chrysanthemi, Paracoccus denitrificans or Rhizobium leguminosarum, the appropriate interposon drug resistance is usually expressed and, compared to the non-mutated plasmid, much reduced levels of C23O activity are detected. Thus, the selection and/or characterization of Ω insertional mutations can be carried out in these bacterial species.