Molecular relationships of degradative plasmids determined by in situ hybridisation of their endonuclease-generated fragments

Summary Plasmid inter-relationships were studied by hybridisation of a radioactively labelled DNA probe to endonuclease-derived fragmentation patterns of plasmids bound to a nitrocellulose filter. The degradative plasmids SAL and NAH were found to be very closely related, but probably one did not give rise to the other by just a single deletion or insertion. Relationships between SAL and other degradative plasmids are complex; substantial homology was found with TOL and other plasmids encoding toluate dissimilation and significant homology was found with OCT.     

Plasmid characteristics of naphthalene and salicylate biodegradation in Pseudomonas putida

The object of this work was to study the physico-chemical and biological properties of DNAs of the biodegradation plasmids NAH and SAL. A comparative analysis of the physico-chemical parameters for these DNAs made it possible to detect a number of identical properties in them: the same sedimentation profile for covalently-closed circular DNA forms, 68--70 S; the molecular weight of ca. 50 MD; a roughly equal number of fragments (up to 23) was found when the DNAs of NAH and SAL were restricted by EcoRI endonuclease. The transformation of the plasmidless strain PpGI was done.     

Electron microscope heteroduplex mapping of naphthalene oxidation genes on the NAH7 and SAL1 plasmids

Introduction of the transposon Tn5 to serve as a marker allows electron microscope heteroduplex mapping of the naphthalene oxidation genes on the approximately 83-kb NAH7 and the related approximately 85-kb SAL1 plasmids. The electron microscope-mapped gene positions on the NAH7 plasmid are in close agreement with those mapped previously by restriction digestion. The SAL1 plasmid can be considered as a mutant NAH7 plasmid which fails to direct the conversion of naphthalene to salicylate because of a mutational block but retains intact coding sequences for salicylate oxidation. Analysis of heteroduplex molecules formed between the SAL1 and NAH7::Tn5 EcoRI fragments and the known NAH7/SAL1 homology strongly suggest that the SAL1 DNA is completely homologous to NAH7 DNA except that a approximately 2.5-kb DNA segment constituting most of the nahA gene is replaced by approximately 4.6-kb nonhomologous DNA.     

A comparative study of the NAH and TOL catabolic plasmids in Pseudomonas putida

A comparative study of the NAH and TOL catabolic plasmids was carried out to provide information for future genetic manipulation experiments involving these two plasmids. The plasmids were studied in a strain of P. putida and its mutant derivatives. The NAH and TOL plasmids were found to be incompatible. Under the conditions used in these experiments the TOL plasmid transferred into some strains into which NAH was unable to transfer. The use of mutants to remove certain catabolic activities encoded by the bacterial host cell facilitated the allocation of growth genotypes to the NAH and TOL plasmids. TOL encoded the degradation of benzoate, m-toluate and p-toluate, whereas NAH encoded the degradation of naphthalene and salicylate. The other plasmid-associated growth phenotypes were partly plasmid-specified and partly specified by the host cell. The pH optimum of the catechol 2,3-dioxygenase specified by the TOL plasmid was approximately 6.7, whereas that of the NAH-encoded enzyme was approximately 8.3.     

Evolutionary relationships between catabolic pathways for aromatics: Conservation of gene order and nucleotide sequences of catechol oxidation genes of pWW0 and NAH7 plasmids

Summary TOL plasmid pWW0 and plasmid NAH7 encode catabolic enzymes required for oxidative degradation of toluene and naphthalene, respectively. The gene order of the catabolic operon of NAH7 for salicylate oxidation was determined to be: promoter-nahG (the structural gene for salicylate hydroxylase)-nahH (catechol 2,3-dioxygenase)-nahI (hydroxymuconic semialdehyde dehydrogenase)-nahN (hydroxymuconic semialdehyde hydrolase)-nahL (2-oxopent-4-enoate hydratase). This order is identical to that of the isofunctional genes of TOL plasmid pWW0. The complete nucleotide sequence of nahH was determined and compared with that of xylE, the isofunctional gene of TOL plasmid pWW0. There were 20% and 16% differences in their nucleotide and amino acid sequences, respectively. The homology between the NAH7 and TOL pWW0 plasmids ends upstream of the Shine-Dalgarno sequences of nahH and xylE, but the homology continues downstream of these genes. This observation suggested that genes for the catechol oxidative enzymes of NAH7 and TOL pWW0 were derived from a common ancestral sequence which was transferred as a discrete segment of DNA between plasmids.     

Growth characteristics of Pseudomonas strains carrying catabolic plasmids and their cured derivatives

Abstract Cured derivatives of Pseudomonas strains carrying TOL or NAH plasmids were obtained by selection on plates containing benzoic acid at almost the inhibitory concentration. Bacteria containing TOL and NAH plasmids produced deep blue colonies on m -toluate/indole or naphthalene/indole plates and weaker reactions on benzoate/indole plates. The indigo reaction of the TOL strains was ascribed to the plasmid-coded benzoate/toluate oxidase. Strains that had been cured of their plasmids did not produce indigo.     

Isolation of metabolic plasmid DNA from Pseudomonas putida

Metabolic plasmids conferring on $\text{Pseudomonas putida}$ the aromatic growth phenotypes naphthalene, Nah⁺, salicylate, Sal⁺, or toluate, Tol⁺, have been isolated as covalently closed circular DNA in 100 μg amounts. Plasmid DNA was banded in CsCl-ethidium bromide density gradients and sedimentation rates measured in sucrose gradients and by analytical centrifuge. The plasmid sizes found, in millions, were /NAH 42, /SAL 43, /TOL 55, 42. Transformation of metabolic plasmid free $\text{P. putida}$ with the isolated DNA confirmed the respective aromatic pathway gene contents.     

Enzyme recruitment in vitro: use of cloned genes to extend the range of haloaromatics degraded by Pseudomonas sp. strain B13.

DNA fragments containing the xylD and xylL genes of TOL plasmid pWW0 -161 of Pseudomonas putida, which code for the catabolic enzymes toluate 1,2-dioxygenase and dihydrodihydroxybenzoic acid dehydrogenase, respectively, and the nahG gene of the NAH plasmid NAH7 , which codes for salicylate hydroxylase, were cloned in pBR322 vector plasmid. Deletion and insertion mutagenesis were used to localize these genes with respect to crucial endonuclease cleavage sites. The pBR322-based plasmids were ligated to the broad host range cloning vector pKT231 , or derivatives of it, and the hybrid plasmids were introduced into Pseudomonas sp. B13( WR1 ), a bacterium able to degrade 3-chlorobenzoate but not 4-chlorobenzoate, 3,5- dichlorobenzoate , salicylate, or chlorosalicylates . The cloned xylD gene expanded the catabolic range of WR1 to include 4-chlorobenzoate, whereas the cloned xylD - xylL genes enabled the isolation of derivatives of WR1 that degraded 3-chlorobenzoate, 4-chlorobenzoate, and 3,5- dichlorobenzoate . The cloned nahG gene extended the catabolic range of WR1 to include salicylate and 3-, 4-, and 5- chlorosalicylate .     

Application of DNA-DNA colony hybridization to the detection of catabolic genotypes in environmental samples

The application of preexisting DNA hybridization techniques was investigated for potential in determining populations of specific gene sequences in environmental samples. Cross-hybridizations among two degradative plasmids, TOL and NAH, and two cloning vehicles, pLAFR1 and RSF1010, were determined. The detection limits for the TOL plasmid against a nonhomologous plasmid-bearing bacterial background was ascertained. The colony hybridization technique allowed detection of one colony containing TOL plasmid among 10(6) Escherichia coli colonies of nonhomologous DNA. Comparisons between population estimates derived from growth on selective substrates and from hybridizations were examined. Findings indicated that standard sole carbon source enumeration procedures for degradative populations lead to overestimations due to nonspecific growth of other bacteria on the microcontaminant carbon sources present in the media. Population estimates based on the selective growth of a microcosm population on two aromatic substrates (toluene and naphthalene) and estimates derived from DNA-DNA colony hybridizations, using the TOL or NAH plasmid as a probe, corresponded with estimates of substrate mineralization rates and past exposure to environmental contaminants. The applications of such techniques are hoped to eventually allow enumeration of any specific gene sequences in the environment, including both anabolic and catabolic genes. In addition, this procedure should prove useful in monitoring recombinant DNA clones released into environmental situations.     

Selective enrichment of Pseudomonas spp. defective in catabolism after exposure to halogenated substrates.

Significant selective enrichments of mutants defective in catabolic pathways can be achieved by exposure of pseudomonad cells to halogenated analogs of growth substrates. Between 3 and 95% of viable clones rescued from such enrichments have been defective in specific catabolic pathways. This has been demonstrated for eight different catabolic pathways for aromatic compounds in pseudomonads, in which the genes are located on plasmids or on the chromosome. The plasmid-encoded pathways studied include those for the catabolism of p-cymene (CYM), m- and p-xylenes (TOL), naphthalene (NAH), salicylate (SAL), and 4-methylphthalate (MOP), and the chromosome-encoded pathways include those for p-hydroxybenzoate, monohydric phenols, and p-anisate utilization. The recalcitrance of halogenated compounds may, in part, be explained by these observations, which introduce an as yet not widely recognized factor in assessment of biodegradability of halogenated compounds and their effects on the transformation of the natural substrates.     

Application of DNA-DNA colony hybridization to the detection of catabolic genotypes in environmental samples.

The application of preexisting DNA hybridization techniques was investigated for potential in determining populations of specific gene sequences in environmental samples. Cross-hybridizations among two degradative plasmids, TOL and NAH, and two cloning vehicles, pLAFR1 and RSF1010, were determined. The detection limits for the TOL plasmid against a nonhomologous plasmid-bearing bacterial background was ascertained. The colony hybridization technique allowed detection of one colony containing TOL plasmid among 10(6) Escherichia coli colonies of nonhomologous DNA. Comparisons between population estimates derived from growth on selective substrates and from hybridizations were examined. Findings indicated that standard sole carbon source enumeration procedures for degradative populations lead to overestimations due to nonspecific growth of other bacteria on the microcontaminant carbon sources present in the media. Population estimates based on the selective growth of a microcosm population on two aromatic substrates (toluene and naphthalene) and estimates derived from DNA-DNA colony hybridizations, using the TOL or NAH plasmid as a probe, corresponded with estimates of substrate mineralization rates and past exposure to environmental contaminants. The applications of such techniques are hoped to eventually allow enumeration of any specific gene sequences in the environment, including both anabolic and catabolic genes. In addition, this procedure should prove useful in monitoring recombinant DNA clones released into environmental situations.     

scpA, a new salicylate hydroxylase gene localized in salicylate/caprolactam degradation plasmids

Both caprolactams and salicylate biodegradation by Pseudomonas salicylate/caprolactam degraders are controlled by large conjugative plasmids (SAL/CAP). Some of these plasmids have been assigned to the P-7 incompatibility group. The new salicylate 1-hydroxylase gene (scpA) has been detected in SAL/CAP plasmids and partially sequenced. The scpA gene was equally related to the closest homolog genes nahG (NAH7), salA (P. reinekei MT1), and nahU (pND6-1); however, the identity rate did not exceed 72–74%. The synthesis of salicylate 1-hydroxylase ScpA was not induced by salicylate. This enzyme had wide substrate specificity and exhibited the highest specific activity toward 4-methylsalicylate and nonsubstituted salicylate substrates. Furthermore, conjugative pseudomonads’ plasmids of salicylate degradation without the classical nah2 operon, which harbors only salicylate 1-hydroxylase gene nahU have been described for the first time.     

Nucleotide sequence of xylE from the TOL pDK1 plasmid and structural comparison with isofunctional catechol-2,3-dioxygenase genes from TOL, pWW0 and NAH7.

Detailed restriction and nucleotide sequence analysis of the Pseudomonas putida TOL plasmid pDK1 xylE gene revealed significant homology with isofunctional xylE (81.5%) and nahH (78.0%) genes from the TOL pWW0 and NAH7 plasmids. The highest degrees of nucleotide and apparent amino acid conservation (82.2 and 86.4%, respectively) among all three genes were found to exist within a region comprising 264 nucleotides encoding the C terminus. A comparison of localized regions revealed significantly greater homology between xylEpWW0 and xylEpDK1 within the C-terminal region, whereas xylEpWW0 and nahH showed greater similarity at the N terminus. The possibility that xylEpWW0 may represent a genetic hybrid of xylEpDK1 and nahH is discussed.     

Plasmid-mediated mineralization of naphthalene, phenanthrene, and anthracene.

The well-characterized plasmid-encoded naphthalene degradation pathway in Pseudomonas putida PpG7(NAH7) was used to investigate the role of the NAH plasmid-encoded pathway in mineralizing phenanthrene and anthracene. Three Pseudomonas strains, designated 5R, DFC49, and DFC50, were recovered from a polynuclear aromatic hydrocarbon-degrading inoculum developed from a manufactured gas plant soil slurry reactor. Plasmids pKA1, pKA2, and pKA3, approximately 100 kb in size, were isolated from these strains and characterized. These plasmids have homologous regions of upper and lower NAH7 plasmid catabolic genes. By conjugation experiments, these plasmids, including NAH7, have been shown to encode the genotype for mineralization of [9-14C]phenanthrene and [U-14C]anthracene, as well as [1-14C]naphthalene. One strain, Pseudomonas fluorescens 5RL, which has the complete lower pathway inactivated by transposon insertion in nahG, accumulated a metabolite from phenanthrene and anthracene degradation. This is the first direct evidence to indicate that the NAH plasmid-encoded catabolic genes are involved in degradation of polynuclear aromatic hydrocarbons other than naphthalene.     

SAL-TOL in vivo recombinant plasmid pKF439.

SAL-TOL in vivo recombinant plasmid pKF439 was characterized in a strain from a mixed culture of bacteria harboring various degradative plasmids. Analysis of the gene organization of pKF439 revealed that the 57-kilobase TOL fragment, including the 40-kilobase TOL metabolic region, was inserted into the complete SAL replicon at the position of SmaI-C within XhoI-B of SAL. The molecular size of pKF439 was calculated to be 138 kilobases. pKF439 could be transferred to Pseudomonas putida and Pseudomonas aeruginosa at high frequency, and the transconjugants gained the ability to grow with m-xylene, m-toluate, and salicylate as the sole carbon source.     

Cloning and expression in Escherichia coli of the naphthalene degradation genes from plasmid NAH7

The genes encoding the enzymes responsible for conversion of naphthalene to 2-hydroxymuconic acid (nahA through nahI) are contained on a 25-kilobase EcoRI fragment of an 85-kilobase NAH plasmid of Pseudomonas putida. These genes were cloned into the plasmid vectors pBR322 and RSF1010 to obtain the recombinant plasmids pKGX505 and pKGX511, respectively. To facilitate cloning and analysis, an NAH7 plasmid containing a Tn5 transposon in the salicylate hydroxylase gene (nahG) was used to derive the EcoRI fragment. The genes for naphthalene degradation were expressed at a low level in Escherichia coli strains containing the fragment on the recombinant plasmids pKGX505 or pKGX511. This was shown by the ability of whole cells to convert naphthalene to salicylic acid and by in vitro enzyme assays. The expression of at least two of these genes in E. coli appeared to be regulated by the presence of the inducer salicylic acid. In addition, high-level expression and induction appear to be mediated by an NAH plasmid promoter and a regulatory gene located on the fragment. A restriction endonuclease cleavage map of the cloned fragment was generated, and the map positions of several nah genes were determined by analysis of various subcloned DNA fragments.     

Naturally occurring TOL plasmids in Pseudomonas strains carry either two homologous or two nonhomologous catechol 2,3-oxygenase genes

Structural genes for catechol 2,3-oxygenase (C23O) were cloned from the TOL plasmids pWW5, pWW14, pWW74, pWW84, and pWW88 isolated from Pseudomonas strains of diverse geographical origins. Each pKT230-based C23O+ recombinant plasmid carried a 2.05-kilobase XhoI insert which showed strong homology in Southern hybridizations with the xylE gene from the archetype TOL plasmid pWW0. Fragments were mapped for restriction endonuclease sites and were classified into two closely related groups on the basis of restriction maps. C23O structural genes were located on cloned fragments by a combination of subcloning and site-specific mutagenesis. All five TOL plasmids examined yielded clones whose maps differed from that of xylE of pWW0 by only a single XbaI site, but in addition plasmids pWW5, pWW74, and pWW88 carried a second, homologous C23O gene with seven further restriction site differences. The remaining plasmids, pWW14 and pWW84, carried a second nonhomologous C23O gene related to the second C23O gene (C23OII) of TOL plasmid pWW15 described previously (H. Keil, M. R. Lebens, and P. A. Williams, J. Bacteriol. 163:248-255, 1985). Thus, each naturally occurring TOL plasmid in this study appears to carry genes for two meta cleavage dioxygenases.     

Molecular analyses of conjugative, gentamicin-resistance plasmids from staphylococcal clinical isolates

Gentamicin-resistant Staphylococcus aureus and Staphylococcus epidermidis strains which were isolated from infants with staphylococcal bacteremia were analyzed for the presence of self-transmissible gentamicin-resistance (Gmr) plasmids. Conjugative GMr plasmids of approximately 43.8-63 kilobases (kb) were found in all S. aureus strains. Inter- and intra-species transfer of Gmr plasmids by conjugation was observed from S. aureus to S. aureus and to S. epidermidis recipient strains. However, neither inter- nor intra-species transfer of gentamicin resistance by conjugation was observed with nine out of nine S. epidermidis donor strains which were mated with either S. epidermidis or S. aureus recipient strains. These conjugative Gmr plasmids were unable to comobilize a smaller (15-kb) plasmid present in all but two S. aureus clinical isolates. Many of the conjugative Gmr plasmids also carried genetic determinants for kanamycin, tobramycin, neomycin, and ethidium bromide resistance, and for beta-lactamase synthesis. EcoRI restriction endonuclease digests of the S. aureus Gmr conjugative plasmids revealed three different digestion patterns. Four EcoRI restriction endonuclease digestion fragments of 15, 11.4, 6.3, and 4.6 kb in size were common to all plasmids. These plasmids and conjugative Gmr staphylococcal plasmids from other geographical regions shared restriction digestion fragments of similar molecular weights. DNA hybridization with biotinylated S. aureus plasmid pIZ7814 DNA revealed a high degree of homology among these plasmids. A 50.9-kb plasmid from one of the nonconjugative S. epidermidis clinical isolates showed homology with the probe DNA but lacked a portion of a 6.3-kb fragment which was present in all conjugative plasmids and believed to carry much genetic information for conjugation.     

Physical and functional mapping of two cointegrate plasmids derived from RP4 and TOL plasmid pDK1

Cointegrate plasmids were formed in vivo between the broad-host-range R-plasmid RP4 and two catabolic plasmids derived from Pseudomonas putida HS1. One of these was the wild-type plasmid pDK1 encoding the complete inducible toluene/xylene (TOL) catabolic pathway and one was pDKT1, a deletion derivative of pDK1 selected after growth of HS1 on benzoate and supporting growth on only toluene. The two plasmids formed, pDK2 and pDKT2 respectively, each consisted of a complete RP4 replicon in which was an insert of the parent plasmid DNA respectively 40 and 20 kbp in size. The detailed restriction maps of the two plasmids were determined and many of the catabolic genes were located by subcloning and enzyme assay of recombinant plasmids in Escherichia coli and Pseudomonas hosts. The insert in pDK2 contained both operons of the catabolic pathway, the 'upper pathway' operon (xylCAB) and the meta pathway operon (xylDLEGF(I,J,K)H), and a region identified as having the function of the regulator gene xylS. The insert in pDKT2 contained only the upper pathway operon and the regulatory region. Within each of the three coding regions there was great similarity with the same regions on TOL plasmids pWW0 and pWW53-4 apparent (a) by the same order of the genes, (b) by a similar pattern of restriction sites and (c) by hybridization studies. However, the order and orientations of the three coding regions differed from those previously described for both pWW0 and pWW53-4. The significance of these findings to the evolution of TOL plasmids is discussed.     

萘降解质粒ND1.860和NAH7的DNA同源性研究

通过DNA:DNA杂交技术,用NAH7质粒的全部EcoR Ⅰ片段或ECOR Ⅰ A片段作为~(32)P标记的DNA探针,研究了萘降解质粒ND1.860和NAH7之间的DNA同源性。在ND1.860的9个HindⅢ片段中,5个与NAH7有同源性,其中3个与NAH7的编码萘降解途径的EcoR Ⅰ A片段有同源性。