| apparent fusion of the tol plasmid with the r91 drug resistance plasmid in pseudomonas aeruginosa. | the tol catabolic plasmid was shown to be compatible with the r91 drug resistance plasmid. however, the tol plasmid was extremely unstable in mutant pa03 of p. aeruginosa. by selecting for stabilization of the tol plasmid in pa03 harbouring r91, it was possible to isolate a strain in which markers from both r91 and tol appeared to exist in a single recombinant plasmid. this plasmid, pnd3, encoded resistance to carbenicillin, was able to transfer at the same frequency as the r91 plasmid and encod ... | 1977 | 414707 |
| metabolism of toluene and xylenes by pseudomonas (putida (arvilla) mt-2: evidence for a new function of the tol plasmid. | pseudomonas putida (arvilla) mt-2 carries genes for the catabolism of toluene, m-xylene, and p-xylene on a transmissible plasmid, tol. these compounds are degraded by oxidation of one of the methyl substituents via the corresponding alcohols and aldehydes to benzoate and m- and p-toluates, respectively, which are then further metabolised by the meta pathway, also coded for by the tol plasmid. the specificities of the benzyl alcohol dehydrogenase and the benzaldehyde dehydrogenase for their three ... | 1975 | 1176436 |
| expression and transfer of engineered catabolic pathways harbored by pseudomonas spp. introduced into activated sludge microcosms. | two genetically engineered microorganisms (gems), pseudomonas sp. strain b13 fr1(pfrc20p) (fr120) and pseudomonas putida kt2440(pwwo-eb62) (eb62), were introduced into activated sludge microcosms that had the level of aeration, nutrient makeup, and microbial community structure of activated sludge reactors. fr120 contains an experimentally assembled ortho cleavage route for simultaneous degradation of 3-chlorobenzoate (3cb) and 4-methyl benzoate (4mb); eb62 contains a derivative tol plasmid-enco ... | 1992 | 1444370 |
| analysis of an upstream regulatory sequence required for activation of the regulatory gene xyls in xylene metabolism directed by the tol plasmid of pseudomonas putida. | transcription from the promoter of a positive regulatory gene, xyls, on the tol plasmid of pseudomonas putida is activated by another positive regulator, xylr, in the presence of m-xylene and is dependent on rna polymerase containing the ntra protein (sigma 54). deletion analysis of the upstream region of the xyls gene revealed an upstream regulatory sequence (urs), located between 145 and 188 bp upstream from the transcription start site. the urs is active in either orientation and can be place ... | 1992 | 1620097 |
| substrate-specificity of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase encoded by tol plasmid pww0. metabolic and mechanistic implications. | the substrate-specificities of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase, encoded by tol plasmid pww0 of pseudomonas putida mt-2, were determined. the rates of benzyl alcohol dehydrogenase-catalysed oxidation of substituted benzyl alcohols and reduction of substituted benzaldehydes were independent of the electronic nature of the substituents at positions 3 and 4. substitutions at position 2 of benzyl alcohol affected the reactivity of benzyl alcohol dehydrogenase: the velocity ... | 1992 | 1590768 |
| upstream binding sequences of the xylr activator protein and integration host factor in the xyls gene promoter region of the pseudomonas tol plasmid. | the xylr and xyls genes, which encode the positive regulators of the tol plasmid catabolic pathways, are adjacent genes on the tol plasmid and are transcribed from divergent promoters. transcription from the xyls gene promoter, ps, is positively regulated by effector-activated xylr protein and requires the specific rna polymerase sigma 54 subunit (rpon). deletions and point mutations in the ps upstream region localized the site of xylr interaction to the region between -133 bp and -207 bp (with ... | 1992 | 1579469 |
| degradation of chlorotoluenes by in vivo constructed hybrid strains: problems of enzyme specificity, induction and prevention of meta-pathway. | the activities of the tol plasmid-coded xylene oxygenase, benzylalcohol dehydrogenase, benzaldehyde dehydrogenase of pseudomonas putida strain paw1 were tested with substituted toluenes, benzylalcohols and benzaldehydes, respectively, as substrates. several chlorinated toluenes were shown to induce enzymes of the xylene degradation sequence. conjugative transfer of the tol plasmid from pseudomonas putida strain paw1 to pseudomonas sp. strain b13 and pseudomonas cepacia strain jh230 allowed the i ... | 1992 | 1526468 |
| construction of a cassette enabling regulated gene expression in the presence of aromatic hydrocarbons. | a high-level expression cassette has been constructed from a tol plasmid derived from pseudomonas putida carrying all cis- and trans-acting regulatory elements necessary for transcriptional gene activation in the presence of aromatic hydrocarbons such as toluene. foreign dna can be inserted at unique kpni, saci, and ecori sites 7, 13, and 15 nucleotides downstream of a ribosome binding site. the cassette, flanked by bamhi and ecori restriction sites, was inserted into a broad-host-range vector a ... | 1992 | 1513877 |
| xyls domain interactions can be deduced from intraallelic dominance in double mutants of pseudomonas putida. | the xyls protein is the positive regulator of the tol plasmid-encoded meta-cleavage pathway for the metabolism of alkylbenzoates in pseudomonas putida. this protein is activated by a variety of benzoate analogues. to elucidate the functional domains of the regulator and their interactions, several fusions of the xyls c-terminus to ms2 polymerase and of the n-terminus to beta-galactosidase were constructed but all are inactive. in addition, 15 double mutant xyls genes were constructed in vitro by ... | 1992 | 1465113 |
| construction of a broad host range shuttle vector for gene cloning and expression in actinobacillus pleuropneumoniae and other pasteurellaceae. | we have constructed a pair of broad host range expression vectors, pjff224-nx and pjff224-xn, based on plasmid rsf1010, which enable cloning and efficient expression of genes in actinobacillus pleuropneumoniae and pasteurella haemolytica and in escherichia coli. the vectors consist of the minimal autonomous replicon of the broad host range plasmid rsf1010 and a type ii chloramphenicol acetyl transferase gene for chloramphenicol resistance selection. in addition, they contain a gene expression ca ... | 1992 | 1448612 |
| physiological properties of a pseudomonas strain which grows with p-xylene in a two-phase (organic-aqueous) medium. | pseudomonas putida idaho utilizes toluene, m-xylene, p-xylene, 1,2,4-trimethylbenzene, and 3-ethyltoluene as growth substrates when these hydrocarbons are provided in a two-phase system at 5 to 50% (vol/vol). growth also occurs on luria-bertani medium in the presence of a wide range of organic solvents. the ability of the organism to grow in the presence of organic solvents is correlated with the logarithm of the octanol-water partition coefficient, with dimethyl-phthalate (log p(oct) = 2.3) bei ... | 1992 | 1444381 |
| identification of critical amino-terminal regions of xyls. the positive regulator encoded by the tol plasmid. | the xyls protein is the positive regulator of the promoter controlling the meta-cleavage pathway (pm) for catabolism of certain alkylbenzoates on the tol plasmid of pseudomonas. transcription from pm is mediated by xyls either in the presence of benzoate effectors or through xyls hyperproduction. two regions of the nh2 terminus of xyls (residues 37-45) had been predicted to be involved in effector control of xyls transcriptional activation. different methods were used to induce mutations in this ... | 1992 | 1429638 |
| molecular cloning of the xyll-xyle region from the p. putida tol plasmid, pdk1. | a 5.2 kilobase ecori restriction fragment from the pseudomonas putida hs1 tol plasmid pdk1, encoding a portion of the lower toluene degradation pathway, was cloned into the e. coli plasmid pbr325. a detailed map of the restriction endonuclease sites was constructed and the nucleotide sequence of three contiguous xhoi fragments, with a combined total length of approximately 3.9 kilobases, has been investigated. this region was determined to contain a total of four separate open reading frames, ea ... | 1990 | 1366507 |
| behavior in agricultural soils of a recombinant pseudomonas bacterium that simultaneously degrades alkyl- and haloaromatics. | pseudomonas sp. fr1 (pfrc20p) is a recombinant bacterium able simultaneously to degrade alkyl- and haloaromatics due to the xylxyzl and xyls genes from the tol plasmid borne on the bacterial chromosome, and to the gene encoding for a gamma-lactone isomerase from alcaligenes eutrophus on pfrc20p. the survival of this strain in sterile soils was shown to depend on the physicochemical properties of the soil. the recombinant information was stable in bacteria introduced in soils and conferred select ... | 1992 | 1341985 |
| 4-oxalocrotonate tautomerase, an enzyme composed of 62 amino acid residues per monomer. | the xylh gene encoding 4-oxalocrotonate tautomerase (4-ot) has been located on a subclone of the pseudomonas putida mt-2 tol plasmid pww0 and inserted into an escherichia coli expression vector. several of the genes of the metafission pathway encoded by pww0 have been cloned in e. coli, but the overexpression of their gene products has met with limited success. by utilizing the e. coli alkaline phosphatase promoter (phoa) coupled with the proper positioning of a ribosome-binding region, we are a ... | 1992 | 1339435 |
| purification and characterisation of the nadh:acceptor reductase component of xylene monooxygenase encoded by the tol plasmid pww0 of pseudomonas putida mt-2. | the xylene monooxygenase system encoded by the tol plasmid pww0 of pseudomonas putida catalyses the hydroxylation of a methyl side-chain of toluene and xylenes. genetic studies have suggested that this monooxygenase consists of two different proteins, products of the xyla and xylm genes, which function as an electron-transfer protein and a terminal hydroxylase, respectively. in this study, the electron-transfer component of xylene monooxygenase, the product of xyla, was purified to homogeneity. ... | 1992 | 1327782 |
| a general system to integrate lacz fusions into the chromosomes of gram-negative eubacteria: regulation of the pm promoter of the tol plasmid studied with all controlling elements in monocopy. | a new procedure is described to recombine plasmid-borne lacz fusions into the chromosome of gram-negative eubacteria in order to study promoter activity in monocopy. the procedure is based upon the insertion into the chromosome of a target bacterium of a recombinant transposon that carries dna sequence homology to the regions flanking lacz fusions present in multicopy promotor-probe vectors, which can be mobilized via rp4-mediated transfer but are unable to replicate in non-enteric bacteria. dou ... | 1992 | 1318499 |
| localization and functional analysis of structural and regulatory dehalogenase genes carried on deh from pseudomonas putida pp3. | pseudomonas putida pp3 expressed two dehalogenases, dehi and dehii. the dehi gene (dehi) was located on a mobile dna element (deh) which inserted at high frequencies into target plasmids from its chromosomal location. from a recombinant tol plasmid (pww0) containing a 6.0-kb deh element inserted into the plasmid's 5.6-kb ecori-g restriction endonuclease fragment, an 11.6-kb ecori fragment was cloned. subcloning analysis and insertion mutagenesis produced a structural map of the deh element and l ... | 1992 | 1312534 |
| construction of tracer plasmids for bacillus sphaericus 1593 utilizing the xyle gene from pseudomonas putida. | genetically engineered microorganisms (gems) released into the environment must be traceable in order to accurately assess their impact on the area of release. tracer genes other than those that introduce antibiotic resistance are preferred for use in identifying genetically engineered strains. in this study, we describe the construction of a series of tracer plasmids for use in bacillus sphaericus using the xyle gene from the pseudomonas putida tol plasmid. this gene codes for the enzyme catech ... | 1991 | 2002245 |
| ubiquity of plasmids in coding for toluene and xylene metabolism in soil bacteria: evidence for the existence of new tol plasmids. | thirteen bacteria have been isolated from nine different soil samples by selective enrichment culture on m-toluate (m-methylbenzoate) minimal medium. eight of these were classified as pseudomonas putida, one as a fluorescent pseudomonas sp., and four as nonfluorescent pseudomonas sp. all 13 strains appeared to carry tol plasmids superficially similar to that previously described in p. putida mt-2 in that: (i) all the wild-type strains could utilize toluene, m-xylene, and p-xylene as sole carbon ... | 1976 | 1254555 |
| isolation of a mutant tol plasmid with increased activity and transmissibility from pseudomonas putida (arvilla) mt-2. | strains with greater ability to dissimilate m-toluate were obtained from the wild-type pseudomonas putida (arvilla) mt-2 that harbors the tol plasmid. increased growth of a mutant strain on aromatic substrates was coupled with simultaneous increase in the activity of metapyrocatechase, an enzyme coded by the tol plasmid, without changing its catalytic properties. in the mutant and the wild-type strains, the inducer specificity and the induction kinetics of metapyrocatechase synthesis were the sa ... | 1977 | 830645 |
| regulation of the degradative pathway enzymes coded for by the tol plasmid (pwwo) from pseudomonas putida mt-2. | pseudomonas putida mt-2 carries a plasmid (tol, pwwo) which codes for a single set of enzymes responsible for the catabolism of toluene and m- and p-xylene to central metabolites by way of benzoate and m- and p-toluate, respectively, and subsequently by a meta cleavage pathway. characterization of strains with mutations in structural genes of this pathway demonstrates that the inducers of the enzymes responsible for further degradation of m-toluate include m-xylene, m-methylbenzyl alcohol, and m ... | 1978 | 659369 |
| 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 enco ... | 1977 | 603460 |
| isolation of tol and rp4 recombinants by integrative suppression. | we obtained genetic and molecular evidence of non-thermosensitive recombinants of rp4 (kmr tcr cbr/apr) and the thermosensitive tol plasmid. as first isolated in pseudomonas aeruginosa pao, the recombinant plasmid ptn1 specified noninducible synthesis of tol enzymes and was transmissible to escherichia coli on selection for the transfer of kanamycin resistance. the phenotypic expression of tol genes of ptn1 in e. coli was low and also noninducible. a spontaneous segregant, ptn2, appearing from p ... | 1978 | 418059 |
| tol plasmid in pseudomonas aeruginosa pao: thermosensitivity of self-maintenance and inhibition of host cell growth. | the tol plasmid originally isolated in pseudomonas putida (arvilla) mt-2 was transmissible to strains of the fluorescens group of pseudomonas, i.e., p. putida, p. fluorescens, and p. aeruginosa, except for a strain of p. aeruginosa, strain pao. the same strain, however, could accept the plasmid when its restriction and modification abilities were lost by mutations or by growing at high temperature. in addition, the transmissibility of the tol plasmid from strain pao to p. putida was low when the ... | 1978 | 415040 |
| an endonuclease cleavage map of the plasmid pwwo-8, a derivative of the tol plasmid of pseudomonas putida mt-2. | cleavage sites on the pwwo-8 plasmid were determined for the restriction endonucleases hindiii and xhoi. terminal labelling using dna polymerase i was particularly useful both for the characterisation of the smaller cleavage products and for confirmation of the order of fragments in the intact plasmid. | 1979 | 285316 |
| transposition of plasmid dna segments specifying hydrocarbon degradation and their expression in various microorganisms. | the conjugative tol plasmid (75 mdal), specifying biodegradation of xylenes, toluene, and trimethylbenzene derivatives, undergoes dissociation in pseudomonas aeruginosa pao to a nonconjugative tol(*) plasmid (28 mdal) and a transfer plasmid termed toldelta (48 mdal). the tol(*) plasmid is rendered transmissible through introduction of a number of conjugative plasmids such as factor k, cam, and toldelta but not by the fp2 derivative pr0271. transfer of tol(*) via factor k or toldelta is mediated ... | 1978 | 277912 |
| a cleavage map of the tol plasmid of pseudomonas putida mt-2. | a cleavage map of the tol plasmid pwwo has been determined for the restriction endonucleases hindiii and xhoi. a number of techniques were employed including (i) digestion of purified cleavage products with a second enzyme; (ii) hybridisation of purified xhoi fragments to southern blots of hindiii digest products and (iii) analysis of a number of deletion mutants. | 1979 | 231727 |
| tol is a broad-host-range plasmid. | we readily isolated insertions of the carbenicillin resistance element tn401 into the tol plasmid in pseudomonas putida. hybrid tol::tn401 plasmids stably express the cbr phenotype in pseudomonas aeruginosa and escherichia coli. whereas the replicative and conjugative functions are expressed in both hosts, the ability to grow on m-toluate is only expressed in the pseudomonas species. | 1978 | 97271 |
| characterization of pseudomonas putida mutants unable to catabolize benzoate: cloning and characterization of pseudomonas genes involved in benzoate catabolism and isolation of a chromosomal dna fragment able to substitute for xyls in activation of the tol lower-pathway promoter. | mutants of pseudomonas putida mt-2 that are unable to convert benzoate to catechol were isolated and grouped into two classes: those that did not initiate attack on benzoate and those that accumulated 3,5-cyclohexadiene-1,2-diol-1-carboxylic acid (benzoate diol). the latter mutants, represents by strain pp0201, were shown to lack benzoate diol dehydrogenase (bend) activity. mutants from the former class were presumed either to carry lesions in one or more subunit structural genes of benzoate dio ... | 1992 | 1629155 |
| chromosomal insertion of tol transposons in pseudomonas aeruginosa pao. | insertions of the tol plasmid transposons tn4651 and tn4653 into the pseudomonas aeruginosa pao chromosome were isolated by a temperature selection technique. the locations and orientations of 16 insertions were determined by pulsed field gel electrophoresis and southern hybridization with genomic and tol dna probes. all insertions occurred within a 334 kb region of the chromosome (representing less than 6% of the genome) with nine of the inserts clustered within a 10 kb area. each transposon wa ... | 1991 | 1650814 |
| conjugational transfer of recombinant dna in cultures and in soils: host range of pseudomonas putida tol plasmids. | recombinant tol plasmid pwwo-eb62 allows pseudomonas putida to grow on p-ethylbenzoate. this plasmid can be transferred to other microorganisms, and its catabolic functions for the metabolism of alkylbenzoates are expressed in a limited number of gram-negative bacteria, including members of pseudomonad rrna group i and escherichia coli. transfer of the recombinant plasmid to erwinia chrysanthemi was observed, but transconjugants failed to grow on alkylbenzoates because they lost catabolic functi ... | 1991 | 1660698 |
| 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 gr ... | 1991 | 1672868 |
| cis-diol dehydrogenases encoded by the tol pww0 plasmid xyll gene and the acinetobacter calcoaceticus chromosomal bend gene are members of the short-chain alcohol dehydrogenase superfamily. | in the aerobic degradation of benzoate by bacteria, benzoate is first dihydroxylated by a ring-hydroxylating dioxygenase to form a cis-diol (1,2-dihydroxycyclohexa-3,4-diene carboxylate) which is subsequently transformed to a catechol by an nad(+)-dependent cis-diol dehydrogenase. the structural gene for this dehydrogenase, encoded on tol plasmid pww0 of pseudomonas putida (xyll) and that encoded on the chromosome of acinetobacter calcoaceticus (bend), were sequenced. they encode polypeptides of ... | 1992 | 1740120 |
| biotransformation of nitrobenzene by bacteria containing toluene degradative pathways. | nonpolar nitroaromatic compounds have been considered resistant to attack by oxygenases because of the electron withdrawing properties of the nitro group. we have investigated the ability of seven bacterial strains containing toluene degradative pathways to oxidize nitrobenzene. cultures were induced with toluene vapor prior to incubation with nitrobenzene, and products were identified by high-performance liquid chromatography and gas chromatography-mass spectrometry. pseudomonas cepacia g4 and ... | 1991 | 1781679 |
| duplication of both xyl catabolic operons on tol plasmid pww15. | pseudomonas fluorescens mt15 is the host of the large (250 kbp) tol plasmid pww15. we have shown by a combination of hybridization, molecular cloning and enzyme assay that pww15 carries two distinct regions which share homology with the upper pathway operons (xylcmabn) of other tol plasmids and two distinct regions which are homologous to the meta pathway operons (xylxyzltegfjqkih) of other tol plasmids. both the areas of homology to the upper pathway operons appear to carry all of the structura ... | 1991 | 1791436 |
| dna sequence determination of the tol plasmid (pwwo) xylgfj genes of pseudomonas putida: implications for the evolution of aromatic catabolism. | the meta operon of the pseudomonas putida tol plasmid (pwwo) encodes all enzymes of a meta-cleavage pathway for the metabolism of benzoic acids to krebs-cycle intermediates. we have determined and analysed the nucleic acid sequence of a 3442 bp region of the meta operon containing the xyl-gfj genes whose products are involved in the post meta-ring fission transformation of catechols. homology analysis of the xylgfj gene products revealed evidence of biochemical relatedness, suggested enzymatic m ... | 1991 | 1791759 |
| [localization of camphor degradative plasmids on the chromosome of pseudomonas putida strains paw]. | camphor degradative plasmids (cam, prk1) are preferentially situated on chromosomes of pseudomonas putida strains paw. after having been transferred into cam+ strains, the tol plasmid pwwo dissociates into the cryptic plasmid pwwo-8 and chromosome-borne transposon tn4651. the opposite situation, i.e. reconstruction of the tol plasmid pwwo from the cryptic plasmid pwwo-8 and chromosome-borne catabolic operons of the pwwo plasmid has been described. cam- derivatives of the cam plasmid were obtaine ... | 1991 | 1855659 |
| activation of the pseudomonas tol plasmid upper pathway operon. identification of binding sites for the positive regulator xylr and for integration host factor protein. | expression of the pseudomonas putida tol plasmid upper pathway operon requires a promoter that belongs to the -12/-24 class. stimulation of transcription from this promoter is positively controlled by the effector-activated xylr protein and requires a form of rna-polymerase holoenzyme containing the rpon-encoded sigma factor, sigma 54. xylr-dependent stimulation of transcription from the pseudomonas tol upper pathway promoter was examined using deletions, insertions, and in vivo dimethyl sulfate ... | 1991 | 1874736 |
| mathematical analysis of catabolic function loss in a population of pseudomonas putida mt-2 during non-limited growth on benzoate. | pseudomonas putida mt-2, harbouring the tol plasmid pww0, was grown continuously on benzoate in a phauxostat at a non-limited rate. the gradual decrease in the population carrying the complete tol plasmid was caused predominantly by a growth-rate advantage of spontaneous mutants carrying a partially deleted plasmid (tol- cells). the growth-rate difference (v) was quantified both by measuring the increase in the dilution rate (from 0.68 to 0.79 h-1; v = 0.11 h-1) and by mathematical analysis of t ... | 1991 | 1919510 |
| stability of tol plasmid pww0 in pseudomonas putida mt-2 under non-selective conditions in continuous culture. | pseudomonas putida mt-2, harbouring the tol plasmid pww0, was grown in chemostat culture under succinate-, sulphate-, ammonium- or phosphate-limitation at different dilution rates. the fraction of mutant cells lacking the plasmid-encoded enzymes for the degradation of toluene and xylene (tol- cells), was determined. genetic analysis revealed that all tol- cells isolated harboured partially deleted plasmids, lacking the tol catabolic genes. the growth-rate advantage of the tol- cells was quantifi ... | 1991 | 1919511 |
| comparison of benzyl alcohol dehydrogenases and benzaldehyde dehydrogenases from the benzyl alcohol and mandelate pathways in acinetobacter calcoaceticus and from the tol-plasmid-encoded toluene pathway in pseudomonas putida. n-terminal amino acid sequences, amino acid compositions and immunological cross-reactions. | 1. n-terminal sequences were determined for benzyl alcohol dehydrogenase, benzaldehyde dehydrogenase i and benzaldehyde dehydrogenase ii from acinetobacter calcoaceticus n.c.i.b. 8250, benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase encoded by the tol plasmid pww53 in pseudomonas putida mt53 and yeast k(+)-activated aldehyde dehydrogenase. comprehensive details of the sequence determinations have been deposited as supplementary publication sup 50161 (5 pages) at the british library d ... | 1991 | 1989592 |
| primary structure of xylene monooxygenase: similarities to and differences from the alkane hydroxylation system. | xylene monooxygenase, encoded by the tol plasmid of pseudomonas putida, catalyzes the oxidation of toluene and xylenes and consists of two different subunits encoded by xyla and xylm. in this study, the complete nucleotide sequences of these genes were determined and the amino acid sequences of the xyla and xylm products were deduced. the xylm sequence had a 25% homology with alkane hydroxylase, which catalyzes the omega-hydroxylation of fatty acids and the terminal hydroxylation of alkanes. the ... | 1991 | 1999388 |
| an upstream xylr- and ihf-induced nucleoprotein complex regulates the sigma 54-dependent pu promoter of tol plasmid. | transcription from promoter pu of the upper catabolic operon of the pseudomonas putida tol plasmid which specifies conversion of toluene/xylenes to benzoate/toluates is activated by the tol-encoded regulator xylr protein in the presence of substrates of the catabolic pathway and in conjunction with the sigma 54(ntra)-containing form of rna polymerase. this regulatory circuit was faithfully reproduced in escherichia coli in single copy gene dosage by integrating the corresponding controlling dete ... | 1991 | 2022186 |
| survival in soils of an herbicide-resistant pseudomonas putida strain bearing a recombinant tol plasmid. | pseudomonas putida eez15(pww0-eb62) is a phosphinothricin (ppt)-resistant strain with a recombinant tol plasmid which allows the strain to grow on p-ethylbenzoate. the survival of this strain in sterile agricultural soils depends on the physicochemical properties of the soil. the recombinant pww0-eb62 plasmid and its catabolic functions were stable for periods of up to 1 month in bacteria introduced in unamended soils and only conferred selective advantage to the host bacteria without the plasmi ... | 1991 | 2036014 |
| purification and characterisation of tol plasmid-encoded benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase of pseudomonas putida. | benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase, two enzymes of the xylene degradative pathway encoded by the plasmid tol of a gram-negative bacterium pseudomonas putida, were purified and characterized. benzyl alcohol dehydrogenase catalyses the oxidation of benzyl alcohol to benzaldehyde with the concomitant reduction of nad+; the reaction is reversible. benzaldehyde dehydrogenase catalyses the oxidation of benzaldehyde to benzoic acid with the concomitant reduction of nad+; the re ... | 1990 | 2202600 |
| growth-phase-dependent expression of the pseudomonas putida tol plasmid pww0 catabolic genes. | pseudomonas putida tol plasmid pww0 catabolic genes are clustered into two operons. the first, the upper operon, is controlled by the xylr regulatory gene, whereas the second, the meta operon, is controlled by the xyls regulatory gene. the xyls gene itself is subjected to control by xylr. in this study, we show that the tol catabolic operons were poorly induced in cells growing at the early-exponential-growth phase but strongly induced in cells at late-exponential-growth phase. we constructed fu ... | 1990 | 2254244 |
| construction of hybrid xyle genes between the two duplicate homologous genes from tol plasmid pww53: comparison of the kinetic properties of the gene products. | the two xyle genes for catechol 2,3-oxygenase, encoded by tol plasmid pww53, carry a common sali restriction site within the reading frame. each gene was cut at the sali site and the 5' end of each gene spliced to the 3' end of the other to form hybrid genes, from both of which catalytically active catechol 2,3-oxygenase activities were expressed. the kinetic parameters were determined for the gene products of both the hybrid and the wild-type xyle genes with catechol, 3-methylcatechol and 4-met ... | 1990 | 2262792 |
| promoter-upstream activator sequences are required for expression of the xyls gene and upper-pathway operon on the pseudomonas tol plasmid. | stimulation of transcription from the pseudomonas tol plasmid xyls gene promoter (ps) and the upper-pathway operon promoter (pu) is dependent on the positive regulator protein xylr activated by an effector molecule such as 3-cholorotoluene, and on rpon, an rna polymerase sigma factor. mutational analysis of the ps and pu promoters showed that upstream activator sequences located between -110 and -218bp upstream of the main transcription initiation point are required for regulated expression from ... | 1990 | 2287278 |
| biotransformation of substituted benzoates to the corresponding cis-diols by an engineered strain of pseudomonas oleovorans producing the tol plasmid-specified enzyme toluate-1,2-dioxygenase. | the conversion of substituted benzoates into 1,2-cis-dihydroxycyclohexa-3,5-diene carboxylic acids (cis-diols) was effected by using escherichia coli and pseudomonas recombinants carrying the xylxyz genes originating from the pseudomonas putida mt-2 tol plasmid, thus producing toluate-1,2-dioxygenase. pseudomonas oleovorans gpo12 recombinants readily produced meta- and para-substituted cis-diols, but were limited in their oxidation of ortho-substituted substrates. | 1990 | 2306096 |
| 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 pvi ... | 1990 | 2325624 |
| signal-regulator interactions. genetic analysis of the effector binding site of xyls, the benzoate-activated positive regulator of pseudomonas tol plasmid meta-cleavage pathway operon. | this study reports a genetic analysis of the interactions between a positive regulator of gene expression and its effector molecules. transcription of the tol plasmid meta-cleavage pathway operon is specifically stimulated by the xyls protein positive regulator either through activation of this regulator by benzoate effectors or through its hyperproduction. one xyls mutant that exhibits constitutive expression of the operon promoter has been characterized, together with six mutants encoding alte ... | 1990 | 2407853 |
| genetic, functional and sequence analysis of the xylr and xyls regulatory genes of the tol plasmid pww0. | mutant derivatives of a plasmid, pcf20, which carries the xhoi-d fragment of the tol plasmid pww0 have been isolated using tn5 transposon mutagenesis. insertion mutations of the xylr and xyls regulatory genes of the catabolic pathway have been isolated and characterized and their ability to induce catechol 2,3-oxygenase activity determined. analysis of the insertion mutants and also segments of the xhoi-d fragment cloned into plasmid puc8 in maxicells has identified a 68 kda polypeptide product ... | 1986 | 2430049 |
| expression of the regulatory gene xyls on the tol plasmid is positively controlled by the xylr gene product. | the regulatory gene xyls on the tol plasmid of pseudomonas putida activates the transcription of the xyldlegf operon for the m-toluate-degrading pathway in the presence of m-toluate. the gene also activates the transcription of the same operon in the presence of m-xylene or m-methylbenzyl alcohol, but for this activation another regulatory gene, xylr, is required. in this study we examined the xyls expression by determining the mrna by reverse transcriptase mapping and by monitoring the enzyme a ... | 1987 | 2440045 |
| omega mutagenesis in gram-negative bacteria: a selectable interposon which is strongly polar in a wide range of bacterial species. | we have used the 2.0-kb dna fragment omega [prentki and krisch, gene 29 (1984) 303-313] to mutagenize in vitro a broad-host-range plasmid carrying the entire meta-cleavage pathway of the pseudomonas putida tol plasmid pww0. the mutant plasmids were subsequently introduced by conjugal mobilization into a variety of gram-negative bacteria. the omega fragment carries a selectable marker (aada+; spcr/smr), which is expressed in all species tested, as well as flanking transcription and translation te ... | 1985 | 2998930 |
| a simple procedure for transferring genes cloned in escherichia coli vectors into other gram-negative bacteria: phenotypic analysis and mapping of tol plasmid gene xylk. | a simple method to transfer non-conjugative escherichia coli plasmids to other gram-negative bacteria and their maintenance is described. this method involves generation of inverse transposition-mediated cointegrates of the non-conjugative e. coli plasmid with a conjugative incw broad-host-range plasmid, r388, carrying tn10. isolation of such cointegrates was readily effected by conjugal transfer from an e. coli donor containing the two plasmids to an e. coli recipient, with selection for transc ... | 1989 | 2548929 |
| characterization of five genes in the upper-pathway operon of tol plasmid pww0 from pseudomonas putida and identification of the gene products. | the upper operon of the tol plasmid pww0 of pseudomonas putida encodes a set of enzymes which transform toluene and xylenes to benzoate and toluates. the genetic organization of the operon was characterized by cloning of the upper operon genes into an expression vector and identification of their products in escherichia coli maxicells. this analysis showed that the upper operon contains at least five genes in the order of xylc-xylm-xyla-xylb-xyln. between the promoter of the operon and xylc, the ... | 1989 | 2549010 |
| molecular analysis of a plasmid-encoded phenol hydroxylase from pseudomonas cf600. | pseudomonas strain cf600 is able to utilize phenol and 3,4-dimethylphenol as sole carbon and energy source. we demonstrate that growth on these substrates is by virtue of plasmid-encoded phenol hydroxylase and a meta-cleavage pathway. screening of a genomic bank, with dna from the previously cloned catechol 2,3-dioxygenase gene of the tol plasmid pww0, was used in the identification of a clone which could complement a phenol-hydroxylase-deficient transposon insertion mutant. deletion mapping and ... | 1989 | 2559941 |
| nucleotide sequence and expression of the catechol 2,3-dioxygenase-encoding gene of phenol-catabolizing pseudomonas cf600. | pseudomonas cf600 degrades phenol and some of its methylated derivatives via a plasmid-encoded catabolic pathway. the catechol 2,3-dioxygenase (c23o) enzyme of this pathway catalyses the conversion of catechol to 2-hydroxymuconic semialdehyde. we have determined the nucleotide (nt) sequence of the dmpb structural gene for this enzyme, and expressed and identified its polypeptide product in escherichia coli. the xyle gene of tol plasmid pwwo and the nahh gene of plasmid nah7 encode analogous c23o ... | 1989 | 2620833 |
| involvement of pseudomonas putida rpon sigma factor in regulation of various metabolic functions. | the rpon protein was originally identified in escherichia coli as a sigma (sigma) factor essential for the expression of nitrogen regulons. in the present study we cloned the pseudomonas putida rpon gene and identified its gene product as a protein with an apparent molecular weight of 78,000. a mutant rpon gene was constructed by in vitro insertion mutagenesis with a kanamycin cassette. a p. putida rpon mutant was then isolated by replacement of the intact chromosomal rpon gene by the mutant rpo ... | 1989 | 2666396 |
| divergent evolution of chloroplast-type ferredoxins. | the tol plasmid pww0 of pseudomonas putida encodes a set of enzymes required for the oxidation of toluene to krebs cycle intermediates. the structural genes for these enzymes are encoded in two operons which comprise the xylcmabn and xylxyzltegfjqkih genes, respectively. the function of the xylt gene has not yet been identified. the nucleotide sequence of xylt was determined in this study and putative gene product was shown to contain a sequence characteristic for chloroplast-type ferredoxins. t ... | 1991 | 2065785 |
| [comparative study of aromatic ring meta-cleavage enzymes in pseudomonas strains with plasmid and chromosomal genetic control of the catabolism of biphenyl and m-toluate]. | it was shown that two different enzymes of aromatic ring oxidative meta-cleavage (2,3-dihydroxybiphenyl-1,2-dioxygenase), dbo and catechol-2,3-dioxygenase, c230) function in pseudomonas strains with a plasmid and chromosomal genetic control of biphenyl and toluate catabolism. a comparative analysis of dbo's and c230's expressed by the pbs241 biphenyl degradative plasmid in p. putida bs893, pbs311 in p. putida u83, chromosomal genes in p. putida bf and c230 from p. putida paw160 (pwwo) was carrie ... | 1990 | 2096950 |
| use of a novel cassette to label phenotypically a cryptic plasmid of bacillus subtilis and map loci involved in its stable maintenance. | in order to facilitate studies on the maintenance of cryptic plasmids from gram-positive bacteria we have constructed a novel cassette capg1000 (5.0 kb) which carries both a selectable marker (chloramphenicol resistance from staphylococcus aureus plasmid pc194) and a screenable marker (the xyle gene from the tol plasmid of pseudomonas putida expressed from a cloned promoter of bacillus phage spo2) and which is flanked by terminators to prevent transcription from the cassette activating or inhibi ... | 1990 | 2116498 |
| loss of tdn catabolic genes by deletion from and curing of plasmid ptdn1 in pseudomonas putida: rate and mode of loss are substrate and ph dependent. | the ability to degrade aromatic amines and m-toluate (tdn+ phenotype), encoded by plasmid ptdn1, was lost from pseudomonas putida hosts after subculture in benzoate, succinate, acetate and glucose minimal medium, the fastest rate of loss occurring where benzoate was the substrate. tdn- cells had either lost the entire ptdn1 plasmid or suffered a recombinational deletion of a specific 26 kbp region. proportional increase of tdn- cells resulted from their growth-rate advantage, and additionally, w ... | 1990 | 2168928 |
| genetic analysis of chromosomal operons involved in degradation of aromatic hydrocarbons in pseudomonas putida tmb. | the catabolic pathway for the degradation of aromatic hydrocarbons encoded by pseudomonas putida tmb differs from the tol plasmid-encoded pathway as far as regulation of the upper pathway is concerned. we found, by analyzing tn5-induced mutants and by southern blot hybridization with appropriate probes derived from the tol plasmid pww0, that the catabolic genes of strain tmb were located on the bacterial chromosome and not on the 84-kb plasmid harbored by this strain. the catabolic genes of tmb ... | 1990 | 2172213 |
| upstream regulatory sequence for transcriptional activator xylr in the first operon of xylene metabolism on the tol plasmid. | transcription of the first operon coding for m-xylene-degrading enzymes on the tol plasmid of pseudomonas putida is activated by the xylr gene product in the presence of m-xylene. the operon has the consensus sequence of the ntr/nif promoters at -24 and -12 regions, and the transcription is dependent on an rna polymerase containing a sigma factor ntra (rpon or sigma 54). deletion derivatives of the upstream sequence of the operon promoter were made in vitro and connected with the xyle gene on a ... | 1990 | 2174974 |
| the meta cleavage operon of tol degradative plasmid pww0 comprises 13 genes. | the meta-cleavage operon of tol plasmid pww0 of pseudomonas putida encodes a set of enzymes which transform benzoate/toluates to krebs cycle intermediates via extradiol (meta-) cleavage of (methyl)catechol. the genetic organization of the operon was characterized by cloning of the meta-cleavage genes into an expression vector and identification of their products in escherichia coli maxicells. this analysis showed that the meta-cleavage operon contains 13 genes whose order and products (in kiloda ... | 1990 | 2183008 |
| a family of positive regulators related to the pseudomonas putida tol plasmid xyls and the escherichia coli arac activators. | the xyls family consists of a least 8 different transcriptional regulators. six of these proteins are positive regulators for the catabolism of carbon sources (benzoate and sugars) in escherichia coli, pseudomonas putida and erwinia carotovora, and two of them are involved in pathogenesis in escherichia coli and yersinia enterocolitica. based on protein alignments, the members of this family exhibit a long stretch of homology at the c-terminal end. the regulators involved in the catabolism of ca ... | 1990 | 2186376 |
| mutations leading to constitutive expression from the tol plasmid meta-cleavage pathway operon are located at the c-terminal end of the positive regulator protein xyls. | the xyls protein is the positive activator of the tol plasmid meta-cleavage pathway operon for the metabolism of alkylbenzoates in pseudomonas putida. the regulator stimulates transcription from the tol meta pathway operon promoter (pm) when activated by benzoate effectors or in the absence of effectors when overproduced. xyls mutant alleles that encode regulators which constitutively mediate expression from pm were isolated and characterized. the mutant proteins all exhibit single amino acid su ... | 1990 | 2193914 |
| cloning and sequence analysis of the ntra (rpon) gene of pseudomonas putida. | the gene encoding a sigma factor ntra (rpon) was cloned from pseudomonas putida by cross-hybridization with a probe containing a part of the corresponding escherichia coli gene. the cloned gene complemented an ntra mutation of e. coli in activation of xyl genes on the tol plasmid. the predicted amino acid (aa) sequence of p. putida ntra (497 aa; mr 56,215) is highly homologous to ntra proteins from azotobacter vinelandii (81.7%), klebsiella pneumoniae (52.6%), and rhizobium meliloti (36.1%). the ... | 1989 | 2695395 |
| degradation of phenol and m-toluate in pseudomonas sp. strain est1001 and its pseudomonas putida transconjugants is determined by a multiplasmid system. | the utilization of phenol, m-toluate, and salicylate (phe+, mtol+, and sal+ characters, respectively) in pseudomonas sp. strain est1001 is determined by the coordinated expression of genes placed in different plasmids, i.e., by a multiplasmid system. the natural multiplasmid strain est1001 is phenotypically unstable. in its phe-, mtol-, and sal- segregants, the plasmid dna underwent structural rearrangements without a marked loss of plasmid dna, and the majority of segregants gave revertants. th ... | 1989 | 2768199 |
| genetic analysis of a transposon carrying toluene degrading genes on a tol plasmid pww0. | toluene degrading (xyl) genes on a pseudomonas tol plasmid pww0 are located within a 39-kb dna portion. the 56-kb region including these xyl genes and its 17-kb derivative with a deletion of the internal 39-kb portion transposed to various sites on target replicons such as pacyc184 and r388 in escherichia coli reca strains. thus the 56- and 17-kb regions were designated tn4651 and tn4652, respectively. genetic analysis of tn4652 demonstrated that its transposition occurs by a two-step process, n ... | 1987 | 2830457 |
| benzoate-dependent induction from the op2 operator-promoter region of the tol plasmid pwwo in the absence of known plasmid regulatory genes. | expression of the lower catabolic pathway of the tol plasmid pwwo requires an aromatic acid inducer and the product of the xyls regulatory gene. pseudomonas putida cells transformed with a plasmid containing the operator-promoter region of the lower pathway (op2 [or pm]), upstream from the catechol 2,3-dioxygenase structural gene, showed enzyme induction in the absence of known tol plasmid regulatory genes. induction was not seen in transformed escherichia coli cells or in a p. putida mutant lac ... | 1988 | 2841300 |
| identification and characterization of tn4653, a transposon covering the toluene transposon tn4651 on tol plasmid pww0. | a pseudomonas tol plasmid pww0 possesses toluene degradative pathway (xyl) genes. unstable maintenance of a pww0 derivative in escherichia coli allowed us to identify two transposable elements each carrying all the xyl genes. one element corresponded to a 56 kb transposon, tn4651, which we had previously characterized. the other element newly identified in this study was 70 kb long, and this element, designated tn4653, completely included tn4651. genetic analysis of tn4653 demonstrated that its ... | 1988 | 2851712 |
| physically associated enzymes produce and metabolize 2-hydroxy-2,4-dienoate, a chemically unstable intermediate formed in catechol metabolism via meta cleavage in pseudomonas putida. | the meta-cleavage pathway of catechol is a major mechanism for degradation of aromatic compounds. in this pathway, the aromatic ring of catechol is cleaved by catechol 2,3-dioxygenase and its product, 2-hydroxymuconic semialdehyde, is further metabolized by either a hydrolytic or dehydrogenative route. in the dehydrogenative route, 2-hydroxymuconic semialdehyde is oxidized to the enol form of 4-oxalocrotonate by a dehydrogenase and then further metabolized to acetaldehyde and pyruvate by the act ... | 1989 | 2681159 |
| regulator and enzyme specificities of the tol plasmid-encoded upper pathway for degradation of aromatic hydrocarbons and expansion of the substrate range of the pathway. | the tol plasmid upper pathway operon encodes enzymes involved in the catabolism of aromatic hydrocarbons such as toluene and xylenes. the regulator of the gene pathway, the xylr protein, exhibits a very broad effector specificity, being able to recognize as effectors not only pathway substrates but also a wide variety of mono- and disubstituted methyl-, ethyl-, and chlorotoluenes, benzyl alcohols, and p-chlorobenzaldehyde. benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase, two upper pa ... | 1989 | 2687253 |
| activation of the xyldlegf promoter of the tol toluene-xylene degradation pathway by overproduction of the xyls regulatory gene product. | the xyls regulatory gene of the pseudomonas putida tol plasmid (pwwo) has been cloned under the transcriptional control of the escherichia coli tac promoter in a broad-host-range controlled-expression vector. induction with isopropylthiogalactoside allowed overproduction and characterization of the xyls product by specific interaction with the tol meta-cleavage pathway operator-promoter region (op2) in vivo in e. coli. examination of plasmid-specified polypeptides in e. coli maxicells led to ide ... | 1987 | 3301806 |
| molecular analysis of regulatory and structural xyl genes of the tol plasmid pww53-4. | pww53-4 is a cointegrate between rp4 and the catabolic plasmid pww53 from pseudomonas putida mt53, which contains 36 kbp of pww53 dna inserted close to the oriv gene of rp4; it encodes the ability to grow on toluene and the xylenes, characteristic of pww53, as well as resistance to tetracycline, kanamycin and carbenicillin, characteristic of rp4. a physical map of the 36 kbp insert of pww53 dna for 11 restriction enzymes is presented, showing that the relative positions of the two xyl operons ar ... | 1987 | 3309179 |
| bacterial metabolism of side chain fluorinated aromatics: cometabolism of 3-trifluoromethyl(tfm)-benzoate by pseudomonas putida (arvilla) mt-2 and rhodococcus rubropertinctus n657. | the tol plasmid-encoded enzymes of the methylbenzoate 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 ... | 1988 | 3365096 |
| transfer and expression of mesophilic plasmid-mediated degradative capacity in a psychrotrophic bacterium. | 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 t ... | 1988 | 3377489 |
| regulatory circuits controlling transcription of tol plasmid operon encoding meta-cleavage pathway for degradation of alkylbenzoates by pseudomonas. | tol plasmid pwwo of pseudomonas putida contains two operons that specify a pathway for the degradation of aromatic hydrocarbons. the 'upper' operon encodes enzymes for the oxidation of toluene to benzoate and xylenes to toluates, whereas the meta-cleavage operon specifies the further oxidation of benzoate and toluates. transcription of the upper pathway operon is positively regulated by the xylr protein, which is activated by toluene/xylenes and their alcohol catabolic products, in combination w ... | 1987 | 3448461 |
| positive regulation and transcription initiation of xyl operons on tol plasmid. | | 1985 | 2990420 |
| determination of the transcription initiation site and identification of the protein product of the regulatory gene xylr for xyl operons on the tol plasmid. | the xylr gene is a regulatory gene on the tol plasmid, which acts in a positive manner on xyl operons for degradation of toluene and xylenes in pseudomonas putida. a dna fragment containing the xylr promoter region was cloned on promoter-probing vectors, and its nucleotide sequence was determined. the transcription initiation site of the xylr gene was determined in cells of p. putida and escherichia coli by s1 nuclease and reverse transcriptase mapping. two initiation sites were detected which w ... | 1985 | 2993247 |
| evolutionary conservation of genes coding for meta pathway enzymes within tol plasmids pww0 and pww53. | pseudomonas putida mt53 contains a tol plasmid, pww53, that encodes toluene-xylene catabolism. pww53 is nonconjugative, is about 105 to 110 kilobase pairs (kbp) in size, and differs significantly in its restriction endonuclease digestion pattern and incompatibility group from the archetypal tol plasmid pww0. an rp4::pww53 cointegrate plasmid, pww53-4, containing about 35 kbp of pww53 dna, including the entire catabolic pathway genes, was formed, and a restriction map for kpni, hindiii, and bamhi ... | 1985 | 2997136 |
| homology between nucleotide sequences of promoter regions of nah and sal operons of nah7 plasmid of pseudomonas putida. | the in vivo transcription start sites of the nah and sal operons of the nah7 plasmid were determined by s1 nuclease mapping and the nucleotide sequence surrounding these transcription start sites was determined. since expression of both of these operons is coordinately controlled by the product of the transcriptional activator gene nahr, the sequences were compared to locate potential sites involved in common regulation. in the 100-base-pair region preceding transcription start sites of both ope ... | 1986 | 3001734 |
| genetic analysis of a relaxed substrate specificity aromatic ring dioxygenase, toluate 1,2-dioxygenase, encoded by tol plasmid pww0 of pseudomonas putida. | toluate 1,2-dioxygenase is the first enzyme of a meta-cleavage pathway for the oxidative catabolism of benzoate and substituted benzoates to krebs cycle intermediates that is specified by tol plasmid pww0 of pseudomonas putida. a collection of derivatives harbouring tn1000 insertions and defective in toluate dioxygenase have been isolated from ppl392, a pbr322-based hybrid plasmid carrying the tol plasmid meta-cleavage pathway operon. in parallel, a series of n-methyl-n'-nitro-n-nitro-soguanidin ... | 1986 | 3010045 |
| nucleotide sequence of a dna segment promoting transcription in pseudomonas putida. | a dna segment that promotes gene expression in pseudomonas putida was identified in ptn8, a mutant plasmid of an rp4-tol recombinant. a promoter on the segment was cloned with a promoter-probe vector containing the xyle gene of the tol plasmid. the xyle gene was expressed under the control of the promoter, and the gene product catechol 2,3-dioxygenase was constitutively synthesized. as analyzed by an s1 nuclease protection assay, the amount of mrna produced in p. putida was more than that in esc ... | 1986 | 3011741 |
| gene order of the tol catabolic plasmid upper pathway operon and oxidation of both toluene and benzyl alcohol by the xyla product. | tol plasmid pww0 specifies enzymes for the oxidative catabolism of toluene and xylenes. the upper pathway converts the aromatic hydrocarbons to aromatic carboxylic acids via corresponding alcohols and aldehydes and involves three enzymes: xylene oxygenase, benzyl alcohol dehydrogenase, and benzaldehyde dehydrogenase. the synthesis of these enzymes is positively regulated by the product of xylr. determination of upper pathway enzyme levels in bacteria carrying tn5 insertion mutant derivatives of ... | 1986 | 3015870 |
| altered effector specificities in regulators of gene expression: tol plasmid xyls mutants and their use to engineer expansion of the range of aromatics degraded by bacteria. | stimulation of transcription from positively regulated promoters involves regulatory proteins that have been activated, generally as a consequence of binding low molecular weight effector molecules. to define essential structural features of effectors for one positively acting gene regulator, the xyls-encoded protein, which activates the tol plasmid meta-cleavage pathway operon promoters, effector activities of a wide range of benzoate derivatives have been systematically analyzed, and mutant xy ... | 1986 | 3022293 |
| nucleotide sequence of the regulatory gene xyls on the pseudomonas putida tol plasmid and identification of the protein product. | the xyls gene is a regulatory gene which positively controls expression of the genes on the tol plasmid for degradation enzymes of benzoate or m-toluate in pseudomonas putida. cloning of the gene in escherichia coli and determination of the nucleotide sequence revealed an open reading frame of 963 bp which corresponds to a protein with an mr of 36,502. the xyls gene was recloned onto a tac-promoter vector, and the product was identified by the maxicell procedure as a protein with an approximate ... | 1986 | 3023186 |
| redesigning metabolic routes: manipulation of tol plasmid pathway for catabolism of alkylbenzoates. | increasing quantities of man-made organic chemicals are released each year into the biosphere. some of these compounds are both toxic and relatively resistant to physical, chemical, or biological degradation, and they thus constitute an environmental burden of considerable magnitude. genetic manipulation of microbial catabolic pathways offers a powerful means by which to accelerate evolution of biodegradative routes through which such compounds might be eliminated from the environment. in the ex ... | 1987 | 3468623 |
| metabolism of benzoate and the methylbenzoates by pseudomonas putida (arvilla) mt-2: evidence for the existence of a tol plasmid. | mutant strains of pseudomonas putida (arvilla) mt-2 which have lost the ability to grow at the expense of m- or p-toluate (methylbenzoate) but retain the ability to grow with benzoate arise spontaneously during growth on benzoate; this genetic loss occurs to a lesser extent during growth on nonaromatic carbon sources in the presence of mitomycin c. the mutants have totally lost the activity of the enzymes of the divergent meta pathway with the possible exception of 2-oxopent-4-enoate hydratase a ... | 1974 | 4418209 |
| the xyls gene positive regulator of tol plasmid pwwo: identification, sequence analysis and overproduction leading to constitutive expression of meta cleavage operon. | the pseudomonas putida tol plasmid pwwo carries an operon that specifies a meta-cleavage pathway for the catabolism of benzoate and toluates whose transcription is positively regulated by the xyls gene product. stimulation of transcription of the operon is thought to result from activation of this protein by pathway substrates/effectors. in the present study, overexpression of the xyls gene has led to identification of the regulator as a 33 kda protein. overexpression of xyls also resulted in pa ... | 1987 | 3475526 |
| 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 bas ... | 1986 | 3023288 |
| gene organization of the first catabolic operon of tol plasmid pww53: production of indigo by the xyla gene product. | the entire operon coding for the enzymes responsible for conversion of toluenes to benzoates has been cloned from tol plasmid pww53 and the position of the genes accurately located. the coding region was 7.4 kilobase pairs (kbp) long, and the gene order was operator-promoter region (op1)-a small open reading frame-xylc (1.6 kbp)-xyla (2.9 kbp)-xylb (1.8 kbp). within the coding region there was considerable homology with the isofunctional region of the archetypal tol plasmid pww0. a central regio ... | 1987 | 3027047 |
| the presence of two complete homologous meta pathway operons on tol plasmid pww53. | pww53 is a 110 kbp catabolic plasmid which encodes the complete pathway for the utilization of toluene and the xylenes. the upper pathway operon xylcab is located between two homologous but distinct meta pathway operons, xyldlegf(i,j,k)h, which are in direct repeat. these have each been cloned on large hindiii restriction fragments ha (17.5 kbp) and hb (15.6 kbp), the restriction sites of which have been mapped. during growth of mt53 on benzoate, mutants which have lost the ability to grow on hy ... | 1988 | 3076178 |
| 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 ... | 1988 | 3076182 |
| broad-host range expression vectors containing manipulated meta-cleavage pathway regulatory elements of the tol plasmid. | the construction of perd20 and perd21, two broad-host range expression vectors, is described. the vectors contain the pm promoter of the meta-cleavage pathway operon of the tol plasmid pwwo; this promoter is present within a polylinker which provides a number of downstream cloning sites close to the transcription initiation site. transcription from the pm promoter in these vectors is controlled not by the natural positive regulator of pm, the xy1s protein, but by an xy1s mutant analogue, xy1s2tr ... | 1988 | 3123271 |
| nucleotide sequence of the regulatory gene xylr of the tol plasmid from pseudomonas putida. | we have determined the nucleotide sequence of the xylr gene for a transcriptional activator for the degradative pathway of aromatic hydrocarbons on the tol plasmid from pseudomonas putida. the 1698-bp sequence for a 566-amino acid (aa) protein (mr 63741) was identified as the xylr-encoding sequence. three regions in xylr show homology to klebsiella pneumoniae ntrc and nifa, both of which are transcriptional activators for the ntr and nif genes involved in the nitrogen metabolism. the central reg ... | 1988 | 3169574 |
| loss of the toluene-xylene catabolic genes of tol plasmid pww0 during growth of pseudomonas putida on benzoate is due to a selective growth advantage of 'cured' segregants. | 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 pww0. experiments with two plasmids identical to pww0 but each with an insert of tn401, which confers resistance to carbenicillin, suggested that the 'benzoate curing' occurs far more frequently by the specific deletion of the 39 kbp region carrying the catabolic genes than by total plasmid loss. t ... | 1988 | 3246596 |