Publications

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
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
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
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
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
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
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
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
conditional-suicide containment system for bacteria which mineralize aromatics.	a model conditional-suicide system to control genetically engineered microorganisms able to degrade substituted benzoates is reported. the system is based on two elements. one element consists of a fusion between the promoter of the pseudomonas putida tol plasmid-encoded meta-cleavage pathway operon (p(m)) and the laci gene encoding lac repressor plus xyls, coding for the positive regulator of p(m). the other element carries a fusion between the p(tac) promoter and the gef gene, which encodes a ...	1991	16348490
nitroaromatics are substrates for the tol plasmid upper-pathway enzymes.	expression of the xylma genes encoding for toluene monoxygenase from the lactose promoter in a broad-host-range plasmid allows the oxidation of toluene and m- and p-nitrotoluene to their corresponding benzyl alcohols and benzaldehydes in pseudomonas putida and escherichia coli. benzyl alcohols accumulate until reaching a concentration of about 80 mum, while benzaldehydes accumulate steadily with time for at least 24 h. tol-encoded benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase recog ...	1992	16348637
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
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
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
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
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
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
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
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
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
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
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 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
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
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
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
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
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
transcriptional control of the pseudomonas putida tol plasmid catabolic pathways.	tol plasmid pww0 of pseudomonas putida contains two operons that specify a pathway for the degradation of aromatic hydrocarbons. the upper pathway operon encodes the enzymes for the oxidation of toluene/xylenes to benzoate/toluates, and the metacleavage pathway operon encodes the enzymes for the further oxidation of these compounds to krebs cycle intermediates. their expression is controlled by the gene products of two divergently transcribed regulatory genes, xyir and xyis. the xyir protein, wh ...	1993	7934920
mineralization of p-methyl-14c-benzoate in soils by pseudomonas putida (pww0).	pseudomonas putida bearing the archetypal tol plasmid pww0 metabolizes p-methylbenzoate through a meta-cleavage pathway. in complex environments such as soils that are relatively rich in organic matter, we observed metabolism of p-methyl-14c-benzoate, which could be monitored as 14co2 evolution from the labelled alkylaromatic. linear 14co2 evolution in soils took place for at least a month, although efficient mineralization of the alkylaromatic required appropriate mass transfer to allow the bac ...	1993	8111534
analysis of the mrna structure of the pseudomonas putida tol meta fission pathway operon around the transcription initiation point, the xylte and the xylfj regions.	the 13 genes encoded by the meta-cleavage operon (approx. 11 kb) of pseudomonas putida tol plasmid pww0 are transcribed from a single promoter, pm. in p. putida, transcription from pm was strictly dependent on the presence of effector-activated xyls protein. three regions of the transcript were analyzed in the wild-type strain of p. putida (pww0) by s1 nuclease protection and primer extension analyses. a major point of transcription initiation was found in the most 5'-end of the operon, which de ...	1993	8241263
the specific growth rate of pseudomonas putida paw1 influences the conjugal transfer rate of the tol plasmid.	the kinetics of the conjugal transfer of a tol plasmid were investigated by using pseudomonas putida paw1 as the donor strain and p. aeruginosa pao 1162 as the recipient strain. short-term batch mating experiments were performed in a nonselective medium, while the evolution of the different cell types was determined by selective plating techniques. the experimental data were analyzed by using a mass action model that describes plasmid transfer kinetics. this method allowed analysis of the mating ...	1993	8250565
new derivatives of tol plasmid pww0.	two new segregants, ppw1-1 and ppw161-1, of pseudomonas putida were isolated from the stock cultures paw85(pww0) and paw85(pww0-161). strain ppw1-1 had lost its ability to grow on m-xylene but was able to grow on m-toluate. a deletion of the left-hand of transposon tn4651, including the upper-operon genes, had taken place in plasmid pww0mut1, isolated from strain ppw1-1. additional deletions were observed in pww0mut1 after 'benzoate-curing' (plasmids pww0mut15, pww0mut19, pww0mut27). the genes o ...	1993	8254307
a substrate-dependent biological containment system for pseudomonas putida based on the escherichia coli gef gene.	a model substrate-dependent suicide system to biologically contain pseudomonas putida kt2440 is reported. the system consists of two elements. one element carries a fusion between a synthetic lac promoter (pa1-04/03) and the gef gene, which encodes a killing function. this element is contained within a transposaseless mini-tn5 transposon so that it can be integrated at random locations on the pseudomonas chromosome. the second element, harbored by plasmid pcc102, is designed to control the first ...	1993	8285679
in vivo reactivation of catechol 2,3-dioxygenase mediated by a chloroplast-type ferredoxin: a bacterial strategy to expand the substrate specificity of aromatic degradative pathways.	the meta-cleavage operon of the tol plasmid pww0 of pseudomonas putida contains 13 genes responsible for the oxidation of benzoate and toluates to krebs cycle intermediates via estradiol (meta) cleavage of (methyl)catechol. the functions of all the genes are known with the exception of xylt. we constructed pww0 mutants defective in the xylt gene, and found that these mutants were not able to grow on p-toluate while they were still capable of growing on benzoate and m-toluate. in the xylt mutants ...	1993	8344270
analysis of pseudomonas gene products using laciq/ptrp-lac plasmids and transposons that confer conditional phenotypes.	novel transposon and plasmid-based broad-host-range expression systems have been developed to facilitate the genetic analysis of gene products of pseudomonas and related gram- bacteria. the properties of laciq/ptrp-lac were used to construct mini-tn5 expression vector transposons and rsf1010-derived plasmids for controlled expression and generation of conditional phenotypes. these plasmids were used to hyper-express the xyls regulator of the meta operon of the tol plasmid of p. putida or the bph ...	1993	8380783
construction of a pseudomonas hybrid strain that mineralizes 2,4,6-trinitrotoluene.	a bacterium, pseudomonas sp. strain c1s1, able to grow on 2,4,6-trinitrotoluene (tnt), 2,4- and 2,6-dinitrotoluene, and 2-nitrotoluene as n sources, was isolated. the bacterium grew at 30 degrees c with fructose as a c source and accumulated nitrite. through batch culture enrichment, we isolated a derivative strain, called pseudomonas sp. clone a, which grew faster on tnt and did not accumulate nitrite in the culture medium. use of tnt by these two strains as an n source involved the successive ...	1993	8468288
a comparison of the multiple alleles of xyls carried by tol plasmids pww53 and pdk1 and its implications for their evolutionary relationship.	both of the independently isolated tol plasmids pww53 and pdk1 contain multiple regions homologous to the xyls regulatory gene of the archetypal tol plasmid pww0. the three homologues on pww53 vary in the extent of their homology to xylspww0, xyls1pww53 is 99% identical to xylspww0 and is located relative to the single copy of xylrpww53 in exactly the same way as xyls and xylr on pww0. the dna sequence of xyls3pww53 is 87% identical to the xylspww0 sequence within the coding region but the non-c ...	1993	8473862
identification of a cis-acting sequence within the pm promoter of the tol plasmid which confers xyls-mediated responsiveness to substituted benzoates.	the dna sequences within the pm promoter/operator region of the meta operon of the tol plasmid of pseudomonas putida, which confer xyls-mediated responsiveness to substituted benzoates, have been identified through deletion analysis and mutagenesis of the region. integrity and proper phasing of two homologous tandem sequences 5'-tgcaapuaapu-pyggnta-3', separated by six base-pairs and overlapping with the -35 region of the pm promoter, was essential for m-toluate activation of a pm-lacz fusion in ...	1993	8478926
kinetic studies on benzyl alcohol dehydrogenase encoded by tol plasmid pwwo. a member of the zinc-containing long chain alcohol dehydrogenase family.	the nucleotide sequence of the structural gene for benzyl alcohol dehydrogenase encoded by tol plasmid pwwo of pseudomonas putida has been determined. benzyl alcohol dehydrogenase is a member of the long-chain zinc alcohol dehydrogenase family and, like other alcohol dehydrogenases of this family, contains two zinc atoms per subunit. benzyl alcohol dehydrogenase, while sharing 31% identical residues with horse liver alcohol dehydrogenase, contains several amino acid substitutions near the active ...	1993	8496150
physical organization of the upper pathway operon promoter of the pseudomonas tol plasmid. sequence and positional requirements for xylr-dependent activation of transcription.	the upper pathway operon of the pseudomonas putida tol plasmid belongs to the -12/-24 class of promoters. these promoters exhibit three regions critical for regulated transcription, namely, the -12/-24 site for rna polymerase/sigma 54 binding, the -55/-67 region for ihf protein binding, and the -130(uas2)/-170(uas1) region, where two sites for xylr binding are located. the xylr-protected g residues located at -131, -139, -160 and -169 were replaced with as, and the activity of the mutant promote ...	1993	8510657
comparison of the nucleotide sequences of the meta-cleavage pathway genes of tol plasmid pww0 from pseudomonas putida with other meta-cleavage genes suggests that both single and multiple nucleotide substitutions contribute to enzyme evolution.	tol plasmid pww0 from pseudomonas putida mt-2 encodes catabolic enzymes required for the oxidation of toluene and xylenes. the structural genes for these catabolic enzymes are clustered into two operons, the xylcmabn operon, which encodes a set of enzymes required for the transformation of toluene/xylenes to benzoate/toluates, and the xylxyzltegfjqkih operon, which encodes a set of enzymes required for the transformation of benzoate/toluates to krebs cycle intermediates. the latter operon can be ...	1993	8510667
carbon source-dependent inhibition of xyl operon expression of the pseudomonas putida tol plasmid.	tol plasmid-encoded degradation of benzyl alcohol by pseudomonas putida is inhibited by glucose and other compounds related to the main carbohydrate metabolism in pseudomonas species. we report here that this effect is exerted at the level of expression of the xyl catabolic operons, and two xyl promoters, pu and ps, were identified as the primary targets of this inhibition. xyl promoter activation was also inhibited by glucose in the heterologous escherichia coli system, apparently not however b ...	1994	8132475
genetic evidence for activation of the positive transcriptional regulator xy1r, a member of the ntrc family of regulators, by effector binding.	the xy1r protein positively controls expression from the pseudomonas putida tol plasmid sigma 54-dependent pu and ps promoters, in response to the presence of aromatic effectors such as m-xylene, m-methylbenzyl alcohol, and p-chlorobenzaldehyde in the culture medium. xy1r also autoregulates its own synthesis. a mutant xy1r regulator called xy1r7 was isolated after nitrosoguanidine mutagenesis of the wild-type gene and phenotypic selection for mutants that had acquired the ability to recognize m- ...	1994	8132529
transcriptional induction kinetics from the promoters of the catabolic pathways of tol plasmid pww0 of pseudomonas putida for metabolism of aromatics.	we determined, under several growth conditions, the kinetics of mrna synthesis from the four pseudomonas putida pww0 plasmid promoters involved in degradation of xylenes and methylbenzyl alcohols via toluates. transcription by xyls of the meta-cleavage pathway operon promoter (pm) for the metabolism of alkylbenzoates was stimulated immediately after the addition of an effector, both in luria-bertani (lb) medium and in minimal medium. activation of the sigma 54-dependent upper-pathway operon prom ...	1994	8169200
genetic evidence that the xyls regulator of the pseudomonas tol meta operon controls the pm promoter through weak dna-protein interactions.	the activation of the pm promoter of the meta operon of the tol plasmid of pseudomonas putida by its cognate xyls activator protein in the presence and absence of benzoate inducers has been examined in specialized escherichia coli strains carrying pm-lacz fusions and the xyls gene in different configurations in which all controlling elements are present in near native conditions and stoichometry. expression of a chromosomal pm-xylx::lacz fusion was primarily dependent on the addition of an effec ...	1994	8195070
analysis of dna bend structure of promoter regulatory regions of xylene-metabolizing genes on the pseudomonas tol plasmid.	the transcription of both the upper operon (op1) coding for m-xylene-degrading enzymes and the positive regulatory gene xyls on the tol plasmid depends on sigma 54-rna polymerase and requires the activator protein xylr that binds to the cis-acting upstream regulatory sequence of each promoter. for transcription of op1 in escherichia coli, integration host factor (ihf) is also required. ihf binds to dna between the upstream regulatory sequence and the promoter sequence of op1. we showed that ihf ...	1994	7896737
substrate specificity of catechol 2,3-dioxygenase encoded by tol plasmid pww0 of pseudomonas putida and its relationship to cell growth.	catechol 2,3-dioxygenase encoded by tol plasmid pww0 of pseudomonas putida consists of four identical subunits, each containing one ferrous ion. the enzyme catalyzes ring cleavage of catechol, 3-methylcatechol, and 4-methylcatechol but shows only weak activity toward 4-ethylcatechol. two mutants of catechol 2,3-dioxygenases (4ecr1 and 4ecr6) able to oxidize 4-ethylcatechol, one mutant (3mcs) which exhibits only weak activity toward 3-methylcatechol but retained the ability to cleave catechol and ...	1994	7928969
metabolic engineering of pseudomonas putida for the simultaneous biodegradation of benzene, toluene, and p-xylene mixture.	for the complete biodegradation of a mixture of benzene, toluene, and p-xylene (btx), a critical metabolic step that can connect two existing metabolic pathways of aromatic compounds (the tod and the tol pathways) was determined. toluate-cis-glycol dehydrogenase in the tol pathway was found to attack benzene-cis-glycol, toluene-cis-glycol, and p-xylene-cis-glycol, which are metabolic intermediates of the tod pathway. based on this observation, a hybrid strain, pseudomonase putida tb101, was cons ...	1994	18615528
fate of pseudomonas putida after release into lake water mesocosms: different survival mechanisms in response to environmental conditions.	to study the fate of pseudomonas putida dsm 3931 in an aquatic environment, cultures of the strain were released into lake water mesocosms. p. putida, bearing the tol-plasmid, was released as a representative xenobiotic-degrading microorganism. the release was carried out in mesocosms with unamended lake water and in lake water with added culture medium to compare the survival of the strain due to the influence of different organic load. as a comparison, the survival of p. putida was followed in ...	1994	24190270
two identical copies of is1246, a 1275 base pair sequence related to other bacterial insertion sequences, enclose the xyl genes on tol plasmid pww0.	two identical direct repeats of a 1275 bp sequence, designated is1246, encompass the xyl genes, which determine the catabolism of toluene, m- and p-xylenes to central metabolites, on the tol catabolic plasmid pww0. is1246 has a terminal inverted repeat of 12 bp (5'gggcacctcgaa3') and contains a major open reading frame of 280 codons. this orf shows significant homology with orfs encoded by a number of bacterial insertion sequences from bacteroides, neisseria and escherichia coli.	1994	7952183
the organization of the pm promoter of the tol plasmid reflects the structure of its cognate activator protein xyls.	the toluate catabolic operon carried by the tol plasmid pww0 of pseudomonas putida is positively regulated by the benzoate-responsive xyls protein which, when activated, stimulates transcription from the operon promoter pm. in this study, the mode in which xyls effects the activity of the pm promoter was examined in vivo by genetic analysis of both protein and promoter variants. substitution of his31asp/ser32pro,leu113pro,phe214leu/glu215a sp/arg216pro or thr312pro, all predicted to disrupt the ...	1994	7969028
loss of the tol meta-cleavage pathway functions of pseudomonas putida strain paw1 (pww0) during growth on toluene.	a derivative of pseudomonas putida strain paw1 bearing the tol plasmid pww0 was isolated from a culture which has grown unlimited on toluene. in contrast to the parent strain paw1, the derivative, strain cg220, is unable to grow with xylenes and toluates, while toluene and benzoate served as substrates. strain cg220 had a remarkable growth advantage against the wild type when grown with toluene. biochemical analysis showed that in strain cg220 toluene was metabolised through the tol plasmid uppe ...	1994	7996396
codon usage patterns suggest independent evolution of two catabolic operons on toluene-degradative plasmid tol pww0 of pseudomonas putida.	tol plasmid pww0 of pseudomonas putida encodes a set of enzymes responsible for the degradation of toluene. the structural genes for these catobolic enzymes are clustered into two operons--namely, the xy/cmab and xy/xyzltegfjqkih operons. we examined the codon usage patterns of these catabolic genes by measuring the codon-usage distances between pairs of these catabolic genes. the codon-usage distance, d, between gene 1 and gene 2 was defined as d = [sigma(pj-qj)2]1/2, are the frequencies of the ...	1994	8007001
transposon-mediated mobilization of chromosomally located catabolic operons of the cam plasmid by tol plasmid transposon tn4652 and cam plasmid transposon tn3614.	the cam (camphor degradation) plasmid is integrated into the chromosome of pseudomonas putida paw-line strains and is not self-transferable as a plasmid via conjugation. our results show that the mobilization of chromosomally located cam and the integration of cam-operons into the chromosome of the new cam+ transconjugants is a reca-independent process mediated by transposons tn4652 (17 kbp) and tn3614 (7.2 kbp). transposon tn3614 is apparently identical to the left-hand and the right-hand seque ...	1994	8012608
functional and structural relationship of various extradiol aromatic ring-cleavage dioxygenases of pseudomonas origin.	the extradiol ring-cleavage dioxygenases derived from seven different pseudomonas strains were expressed in escherichia coli and the substrate specificities were investigated for a variety of catecholic compounds. the substrate range of four 2,3-dihydroxybiphenyl dioxygenases from biphenyl-utilizing bacteria, 3-methylcatechol dioxygenase from toluene utilizing pseudomonas putida f1, 1,2-dihydroxynaphthalene dioxygenase from a nah7 plasmid, and catechol 2,3-dioxygenase from a tol plasmid pww0 wer ...	1994	8020752
chromosomal gene capture mediated by the pseudomonas putida tol catabolic plasmid.	the pseudomonas putida tol plasmid pww0 is able to mediate chromosomal mobilization in the canonical unidirectional way, i.e., from donor to recipient cells, and bidirectionally, i.e., donor-->recipient-->donor (retrotransfer). transconjugants are recipient cells that have received dna from donor cells, whereas retrotransconjugants are donor bacteria that have received dna from a recipient. the tol plasmid pww0 is able to directly mobilize and retromobilize a kanamycin resistance marker integrat ...	1994	8045894
co-regulation by bent dna. functional substitutions of the integration host factor site at sigma 54-dependent promoter pu of the upper-tol operon by intrinsically curved sequences.	the role of integration host factor (ihf) in the regulation of the sigma 54-dependent promoter pu of the tol plasmid of pseudomonas putida has been examined. we have selected in vivo insertions of intrinsically curved dna that restore the responsiveness of an ihf-binding site deletion variant of pu to the cognate activator of the system, xylr. we found five pu derivatives which had inserted a core sequence with 6 phased [a]6 tracts, flanked by different lengths of dna at the location of the form ...	1994	8077217
tol plasmid-specified xylene oxygenase is a wide substrate range monooxygenase capable of olefin epoxidation.	xylene oxygenase, which is encoded on the tol plasmid pwwo of pseudomonas putida mt-2, is a key enzyme system in the degradation of toluene and xylenes by this organism. it was expressed in an escherichia coli recombinant strain carrying the xylma structural genes. this recombinant, which expressed xylene oxygenase from the heat-shock induced lambda pl promoter, was analyzed for its potential as a biocatalytic tool so as to effect the oxidation of side chains of aromatic hydrocarbons to the corr ...	1994	7764991
molecular and biochemical characterization of two meta-cleavage dioxygenases involved in biphenyl and m-xylene degradation by beijerinckia sp. strain b1.	beijerinckia sp. strain b1 is able to grow on either biphenyl or m-xylene as the sole source of carbon and is capable of cooxidizing many polycyclic aromatic hydrocarbons. the catabolic pathways for biphenyl and m-xylene degradation are coinduced and share common downstream enzymatic reactions. the catabolic pathway for biphenyl degradation involves two meta-cleavage steps, one for 2,3-dihydroxybiphenyl and a second for catechol. the catabolic pathway for m-xylene involves one m-cleavage step fo ...	1995	7768806
involvement of ihf protein in expression of the ps promoter of the pseudomonas putida tol plasmid.	regulation of the xyl gene operons of the pseudomonas putida tol plasmid is mediated by the products of the downstream clustered and divergently oriented xylr and xyls regulatory genes. the xylr-xyls intergenic region contains the xylr and xyls promoters pr and ps, respectively. a binding site for the xylr activator protein is located upstream of ps and overlapping pr. dnase i footprint experiments showed that one of these sites, which overlaps the recognition site for xylr activator, as well as ...	1995	7768832
overlapping substrate specificities of benzaldehyde dehydrogenase (the xylc gene product) and 2-hydroxymuconic semialdehyde dehydrogenase (the xylg gene product) encoded by tol plasmid pww0 of pseudomonas putida.	two aldehyde dehydrogenases involved in the degradation of toluene and xylenes, namely, benzaldehyde dehydrogenase and 2-hydroxymuconic semialdehyde dehydrogenase, are encoded by the xylc and xylg genes, respectively, on tol plasmid pww0 of pseudomonas putida. the nucleotide sequence of xylc was determined in this study. a protein exhibiting benzaldehyde dehydrogenase activity had been purified from cells of p. putida (pww0) (j. p. shaw and s. harayama, eur. j. biochem. 191:705-714, 1990); howev ...	1995	7868591
single amino acids changes in the signal receptor domain of xylr resulted in mutants that stimulate transcription in the absence of effectors.	the xylr protein positively controls expression from the pseudomonas putida tol plasmid sigma 54-dependent "upper" pathway operon promoter (pu) and the xyls gene promoter (ps), in response to the presence of aromatic effectors. two mutant xylr regulators able to stimulate transcription from pu and ps in the absence of effectors were isolated. these mutants exhibited single point mutations, namely asp135-->asn and pro85-->ser. both mutations are located in the amino termini domain of xylr, which ...	1995	7890623
identification of functional residues in a 2-hydroxymuconic semialdehyde hydrolase. a new member of the alpha/beta hydrolase-fold family of enzymes which cleaves carbon-carbon bonds.	the 2-hydroxymuconic semialdehyde hydrolase, xylf, of the pseudomonas putida tol plasmid-encoded pathway for the catabolism of toluene and xylenes, catalyzes one of the rarest types of enzyme reaction (ec 3.7.1.9), the hydrolysis of a carbon-carbon bond in its substrate, the ring-fission product of 3-alkyl-substituted catechols. in this study, amino acid sequence comparisons between xylf and other hydrolases, and analysis of the similarity between the predicted secondary structure of xylf and th ...	1995	7890778
construction and behavior of biologically contained bacteria for environmental applications in bioremediation.	the survival of microorganisms can be predicted through the use of active biological containment systems. we have constructed contained pseudomonas putida strains that degrade alkylbenzoates. the modified strain carries a fusion of the plac promoter to the gef gene, which encodes a killing protein. expression from plac is controlled through a regulatory cascade, so that plac is switched on or off by the absence or presence of alkylbenzoates, respectively. similar uncontained strains were also co ...	1995	7487030
the 4-hydroxy-2-oxovalerate aldolase and acetaldehyde dehydrogenase (acylating) encoded by the nahm and naho genes of the naphthalene catabolic plasmid pww60-22 provide further evidence of conservation of meta-cleavage pathway gene sequences.	we report the complete nucleotide sequence and over-expression of the nahom genes for the acetaldehyde dehydrogenase (acylating) and the 4-hydroxy-2-oxovalerate aldolase from the meta pathway operon of the naphthalene catabolic plasmid pww60-22 from pseudomonas sp. ncimb9816. additional partial sequence analysis of adjacent dna shows the gene order within the operon to be nahnlomk, identical to the order found for the isofunctional genes in the meta pathway operons in the toluene/xylene pathway ...	1995	7496535
quantification of the effect of substrate concentration on the conjugal transfer rate of the tol plasmid in short-term batch mating experiments.	batch mating experiments with pseudomonas putida paw 1 (tol) as a donor and pseudomonas aeruginosa pao 1162 as a recipient strain were performed to quantify the effect of the substrate concentration in the mating medium on the observed plasmid transfer rate coefficient. the impact of the substrate concentration in the mating medium was highly correlated with the growth history of the donor strain. when the donor strain was harvested in exponential growth phase, no impact was observed; when the d ...	1995	7576502
the sigma 54-dependent promoter ps of the tol plasmid of pseudomonas putida requires hu for transcriptional activation in vivo by xylr.	in the presence of toluene and xylenes, the sigma 54-dependent ps promoter of the tol (toluene biodegradation) plasmid pww0 of pseudomonas putida is activated at a distance by the xylr protein, of the ntrc family of transcriptional regulators. since contacts between xylr bound to upstream activating sites and the rna polymerase require the looping out of the intervening dna segment, the intrinsic curvature, the bendability of the corresponding sequence, and the spatial effects of protein-induced ...	1995	7601841
isolation and expansion of the catabolic potential of a pseudomonas putida strain able to grow in the presence of high concentrations of aromatic hydrocarbons.	pseudomonas putida dot-t1 was isolated after enrichment on minimal medium with 1% (vol/vol) toluene as the sole c source. the strain was able to grow in the presence of 90% (vol/vol) toluene and was tolerant to organic solvents whose log p(ow) (octanol/water partition coefficient) was higher than 2.3. solvent tolerance was inducible, as bacteria grown in the absence of toluene required an adaptation period before growth restarted. mg2+ ions in the culture medium improved solvent tolerance. elect ...	1995	7608060
biosensing of benzene derivatives in the environment by luminescent escherichia coli.	sensitive and convenient biosensing of environmental pollutants has been developed by fusing a gene of firefly luciferase to the tol plasmid. tol plasmid of pseudomonas putida encodes a series of enzymes for degradation of benzene and its derivatives. the expression of these enzymes is controlled with the regulating proteins xylr and xyls, whose promoters are activated in the presence of aromatic compounds. the structural gene of firefly luciferase, as a reporter enzyme, was inserted under the c ...	1995	7612210
overexpression of pseudomonas putida catechol 2,3-dioxygenase with high specific activity by genetically engineered escherichia coli.	the cloned xyle gene encoding catechol 2,3-dioxygenase (metapyrocatechase) from tol plasmid in pseudomonas putida mt-2 has been expressed in escherichia coli w3110 to a level of approximately 15% of the total soluble protein. of the total iron in the crude extract, 45% was on the enzyme. the crystallized enzyme from e. coli had higher iron content (3.7 mol/mol enzyme) and specific activity (536 u/mg) than the enzyme from p. putida mt-2. however, no differences were observed in physicochemical, p ...	1995	7629031
integration host factor suppresses promiscuous activation of the sigma 54-dependent promoter pu of pseudomonas putida.	in the presence of m-xylene, the pu promoter of the tol plasmid of pseudomonas putida is activated by the prokaryotic enhancer-binding protein xylr. the intervening dna segment between the upstream activating sequences (uass) and those for rna polymerase binding contains an integration host factor (ihf) attachment site that is required for full transcriptional activity. in the absence of ihf, the pu promoter can be cross-activated by other members of the sigma 54-dependent family of regulatory p ...	1995	7638181
cloning and sequencing of the catechol 2,3-dioxygenase gene of alcaligenes sp. kf711.	the catechol 2,3-dioxygenase is an aromatic ring-fission enzyme catalyzing the conversion of catechol to 2-hydroxymuconic semialdehyde. a catechol 2,3-dioxygenase gene has been cloned from chromosomal dna of alcaligenes sp. kf711, and its sequence was determined. the catechol 2,3-dioxygenase gene was consisted of 927 nucleotides with atg initiation codon and tga termination codon, which can encode a polypeptide of molecular weight 35 kda containing 308 amino acid residues. g+c content of the gen ...	1995	7702624
tetrameric structure and cellular location of catechol 2,3-dioxygenase.	catechol 2,3-dioxygenase from the meta-cleavage pathway encoded on the tol plasmid of pseudomonas putida (pwwo) was investigated by electron microscopy. negatively stained samples of the purified catechol 2,3-dioxygenase revealed that the enzyme consists of four subunits arranged in a tetrahedral conformation. monoclonal antibodies raised against catechol 2,3-dioxygenase showed highly specific reactions and were used to localize the enzyme in escherichia coli (paw31) and p. putida (pwwo), using ...	1995	7710322
substrate specificity differences between two catechol 2,3-dioxygenases encoded by the tol and nah plasmids from pseudomonas putida.	the substrate specificities of two catechol 2,3-dioxygenases, one encoded by xyle on the tol plasmid pww0 and the other encoded by nahh on the nah7 plasmid, were investigated. the xyle catechol 2,3-dioxygenase catalyzes the ring-cleavage of catechol, 3-methylcatechol and 4-methylcatechol. the nahh catechol 2,3-dioxygenase was partially deficient in oxidizing 3-methylcatechol due to defects in two catalytic properties. first, nahh has a lower kcat value for 3-methylcatechol compared to xyle, and ...	1995	7744021
amplification of toluene dioxygenase genes in a hybrid pseudomonas strain to enhance the biodegradation of benzene, toluene, and p-xylene mixture.	a hybrid metabolic pathway through which benzene, toluene, and p-xylene (btx) mixture could be simultaneously mineralized was previously constructed in pseudomonas putida tb101 (lee, roh, kim, biotechnol. bioeng 43: 1146-1152, 1994). in this work, we improved the performance of the hybrid pathway by cloning the todc1c2ba genes in the broad-host-range multicopy vector rsf1010 and by introducing the resulting plasmid ptol037 into p. putida mt-2 which harbors the archetypal tol plasmid. as a result ...	1995	18623248
impact of growth in benzoate and m-toluate liquid media on culturability of pseudomonas putida on benzoate and m-toluate plates.	pseudomonas putida grown in continuous culture on benzoate or m-toluate lost the ability to grow on benzoate or m-toluate plates. a similar effect was not seen with a glucose continuous culture. cells carrying and expressing a tol plasmid rapidly lost their ability to grow on benzoate solid medium.	1995	16535111
role of sigma s in transcription from the positively controlled pm promoter of the tol plasmid of pseudomonas putida.	transcription from the tol plasmid pm promoter is dependent on the xyls regulator activated by benzoate effectors. we analysed transcription from pm in several backgrounds with differing escherichia coli alpha and sigma subunits of rna polymerase. in different rpoa backgrounds, transcription from pm was as high as in the wild-type background throughout the growth curve. in the sigma s-deficient background provided by e. coli rh90, high levels of transcription from pm (xyls/3-methylbenzoate depen ...	1995	8825089