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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.1979285316
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 ...1977830645
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 ...19921312534
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 ...19921629155
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 ...19911672868
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 ...19911999388
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 ...19902172213
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 ...19852993247
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 ...19863001734
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 ...19863011741
comparison of the meta pathway operons on nah plasmid pww60-22 and tol plasmid pww53-4 and its evolutionary significance.the regulated meta pathway operon for the catabolism of salicylate on the naphthalene plasmid pww60-22 was cloned into the broad-host-range vector pkt230 on a 17.5 kbp bamhi fragment. the recombinant plasmid conferred the ability to grow on salicylate when mobilized into plasmid-free pseudomonas putida paw130. a detailed restriction map of the insert was derived and the locations of some of the genes were determined by subcloning and assaying for their gene products in escherichia coli and p. pu ...19883254935
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 ...19883365096
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 ...19883377489
the xylabc promoter from the pseudomonas putida tol plasmid is activated by nitrogen regulatory genes in escherichia coli.the xylabc promoter (op1), located on the tol plasmid of pseudomonas putida contains sequences homologous to the conserved regions found in nitrogen fixation (nif) promoters and in other promoters subject to nitrogen control. xyla-lac fusions were constructed in order to monitor expression from the op1 promoter in escherichia coli. transcription was activated in the presence of the heterologous regulatory genes ntrc or nifa from klebsiella pneumoniae as well as by the homologous p. putida regula ...19863520241
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 ...19744418209
nucleotide sequence of the promoter region of the xyldegf operon on tol plasmid of pseudomonas putida.the transcription initiation site of the xyldegf operon on the tol plasmid of pseudomonas putida mt-2 was determined in p. putida and in escherichia coli by s1 nuclease and reverse transcriptase mapping. the induced synthesis of mrna started at the same start point in both p. putida and e. coli, although the amount of mrna in e. coli cells was less than that in p. putida. the nucleotide sequence of the region surrounding the start point was also determined. the ribosome-binding site (rbs) comple ...19846092237
enzyme recruitment in vitro: use of cloned genes to extend the range of haloaromatics degraded by pseudomonas sp. strain b13.dna fragments containing the xyld and xyll genes of tol plasmid pww0 -161 of pseudomonas putida, which code for the catabolic enzymes toluate 1,2-dioxygenase and dihydrodihydroxybenzoic acid dehydrogenase, respectively, and the nahg gene of the nah plasmid nah7 , which codes for salicylate hydroxylase, were cloned in pbr322 vector plasmid. deletion and insertion mutagenesis were used to localize these genes with respect to crucial endonuclease cleavage sites. the pbr322-based plasmids were ligat ...19846327621
chromogenic identification of genetic regulatory signals in bacillus subtilis based on expression of a cloned pseudomonas gene.a method to isolate fragments of dna that promote gene expression in bacillus subtilis is described. the system is based on production of catechol 2,3-dioxygenase [cato2ase; catechol:oxygen 2,3-oxidoreductase (decyclizing), ec 1.13.11.2] encoded by the pseudomonas putida tol plasmid gene xyle. the gene was transferred to ab. subtilis/escherichia coli plasmid vector to construct ptg402. although xyle is functionally expressed in e. coli, cato2ase is not detected in b. subtilis unless a fragment o ...19836405380
metabolism of allylglycine and cis-crotylglycine by pseudomonas putida (arvilla) mt-2 harboring a tol plasmid.spontaneous mutants which acquired the ability to utilize d-allylglycine (d-2-amino-4-pentenoic acid) and dl-cis-crotylglycine (dl-2-amino-cis-4-hexenoic acid) but not l-allylglycine or dl-trans-crotylglycine could be readily isolated from pseudomonas putida mt-2 (pam1). derivative strains of pam1 putatively cured of the tol (pwwo) plasmid were incapable of forming mutants able to utilize the amino acids for growth; however, this ability could be regained by conjugative transfer of the tol (pwwo ...19817287632
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 ...19957768832
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 ...19937934920
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.19947952183
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- ...19948132529
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 ...19938250565
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 ...19938478926
expression of the tol plasmid xyls gene in pseudomonas putida occurs from a alpha 70-dependent promoter or from alpha 70- and alpha 54-dependent tandem promoters according to the compound used for growth.growth of pseudomonas putida (pwwo) on alkylbenzoates requires the expression of the meta pathway operon, which is mediated by the xyls protein after binding of a benzoate effector. alternatively, in cells growing on toluene or its aromatic alcohols, overexpression of xyls mediated by xylr activated by these compounds leads to overproduction of the xyls regulator, which even in the absence of benzoate effectors stimulates transcription from the meta cleavage pathway operon promoter. we show here ...19968636038
structure of catechol 2,3-dioxygenase gene encoded in chromosomal dna of pseudomonas putida kf715.a catechol 2,3-dioxygenase gene in chromosomal dna of p. putida kf715 was cloned and its nucleotide sequence analyzed. the enzyme gene was composed of 924 base pairs with atg initiation codon and tga termination codon, which can encode a polypeptide of molecular weight 35 kda containing 307 amino acids. a promoter-like sequence and a ribosome-binding sequence were identified upstream the enzyme gene. a deduced amino acid sequence of the catechol 2,3-dioxygenase exhibited 94% identity with that o ...19968713131
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 ...19958825089
coactivation in vitro of the sigma54-dependent promoter pu of the tol plasmid of pseudomonas putida by hu and the mammalian hmg-1 protein.the mechanism by which the prokaryotic histone-like protein hu replaces the integration host factor (ihf) in the coactivation of the sigma54-dependent promoter pu of pseudomonas putida has been investigated. by using a preactivated form of the cognate activator protein xylr, we show that the functional replacement of ihf with hu previously suggested in vivo can be faithfully reproduced in vitro with purified components. furthermore, the coactivation effect of ihf on pu could be mimicked not only ...19979098077
transcriptional control of the multiple catabolic pathways encoded on the tol plasmid pww53 of pseudomonas putida mt53.the tol plasmid pww53 encodes a catabolic pathway for the metabolism of toluene. it bears an upper-pathway operon for the oxidation of toluene to benzoate and a copy of the gene that encodes regulatory protein xylr. for metabolism of the aromatic carboxylic acid, it bears two functional homologous meta-pathway operons, together with two functional copies of the xyls regulatory gene (xyls1 and xyls3). in cells growing in the absence of pathway substrates, no mrna from upper- and meta-pathway oper ...19979260942
cloning and sequence analysis of a catechol 2,3-dioxygenase gene from the nitrobenzene-degrading strain comamonas sp js765.comamonas sp strain js765 utilizes nitrobenzene as a carbon and nitrogen source. the initial attack on nitrobenzene is carried out by nitrobenzene 1,2-dioxygenase, which converts nitrobenzene to an unstable nitrohydrodiol that spontaneously decomposes to form catechol and nitrite. catechol is then degraded via a meta cleavage pathway. we now report the cloning of a dna fragment carrying a catechol 2,3-dioxygenase gene from js765. nucleotide sequence analysis revealed three open reading frames (o ...19979451836
development and testing of a bacterial biosensor for toluene-based environmental contaminants.a bacterial biosensor for benzene, toluene, and similar compounds has been constructed, characterized, and field tested on contaminated water and soil. the biosensor is based on a plasmid incorporating the transcriptional activator xylr from the tol plasmid of pseudomonas putida mt-2. the xylr protein binds a subset of toluene-like compounds and activates transcription at its promoter, pu. a reporter plasmid was constructed by placing the luc gene for firefly luciferase under the control of xylr ...19989501440
proton transfer in benzyl alcohol dehydrogenase during catalysis: alternate proton-relay routes.his51 in horse liver alcohol dehydrogenase (adhe) has been proposed to act as a proton donor/acceptor in the nad+/nadh-dependent oxidation/reduction of alcohol/aldehyde. the residue corresponding to his51 of adhe is val51 (val45 in the protein sequence) in benzyl alcohol dehydrogenase (badh) encoded by tol plasmid pww0. the 3-d structure of badh modeled from the crystal structure of adhe suggests that his47 (his41 in the protein sequence, corresponding to arg47 in adhe) of badh would play the ro ...19989521650
establishment of new genetic traits in a microbial biofilm community.conjugational transfer of the tol plasmid (pwwo) was analyzed in a flow chamber biofilm community engaged in benzyl alcohol degradation. the community consisted of three species, pseudomonas putida ri, acinetobacter sp. strain c6, and an unidentified isolate, d8. only p. putida ri could act as a recipient for the tol plasmid. cells carrying a chromosomally integrated laciq gene and a lacp-gfp-tagged version of the tol plasmid were introduced as donor strains in the biofilm community after its fo ...19989603843
critical nucleotides in the upstream region of the xyls-dependent tol meta-cleavage pathway operon promoter as deduced from analysis of mutants.the pm promoter, dependent on tol plasmid xyls regulator, which is activated by benzoate effectors, drives transcription of the meta-cleavage pathway for the metabolism of alkylbenzoates. this promoter is unique in that in vivo transcription is mediated by rna-polymerase with different sigma factors. in vivo footprinting analysis shows that xyls interacted with nucleotides in the -40 to -70 region. in vivo and in vitro methylation of pm shows extensive methylation of t at position -42 in the bot ...19999890992
functional domains of the tol plasmid transcription factor xyls.the alkylbenzoate degradation genes of pseudomonas putida tol plasmid are positively regulated by xyls, an arac family protein, in a benzoate-dependent manner. in this study, we used deletion mutants and hybrid proteins to identify which parts of xyls are responsible for the dna binding, transcriptional activation, and benzoate inducibility. we found that a 112-residue c-terminal fragment of xyls binds specifically to the pm operator in vitro, protects this sequence from dnase i digestion identi ...200010648539
effects of iron limitation on the degradation of toluene by pseudomonas strains carrying the tol (pwwo) plasmid.most aerobic biodegradation pathways for hydrocarbons involve iron-containing oxygenases. in iron-limited environments, such as the rhizosphere, this may influence the rate of degradation of hydrocarbon pollutants. we investigated the effects of iron limitation on the degradation of toluene by pseudomonas putida mt2 and the transconjugant rhizosphere bacterium p. putida wcs358(pwwo), both of which contain the pwwo (tol) plasmid that harbors the genes for toluene degradation. the results of conti ...200111472911
the tol plasmid pww0 xyln gene product from pseudomonas putida is involved in m-xylene uptake.the upper operon of the tol plasmid pww0 of pseudomonas putida encodes a set of enzymes involved in the conversion of toluene and xylenes to their carboxylic acid derivatives. the last gene of the upper operon, xyln, encodes a 465-amino-acid polypeptide which exhibits significant sequence similarity to fadl, an outer membrane protein involved in fatty acid transport in escherichia coli. to analyze the role of the xyln gene product, xyln on tol plasmid pww0 was disrupted by inserting a kanamycin ...200111673437
a la carte transcriptional regulators: unlocking responses of the prokaryotic enhancer-binding protein xylr to non-natural effectors.to investigate the activation mechanism of the enhancer-binding protein xylr encoded by the tol plasmid of pseudomonas putida mt-2, a combinatorial library was generated composed of shuffled n-terminal a domains of the homologous regulators dmpr, xylr and tbut, reassembled within the xylr structure. when the library was screened in vivo for responsiveness to non-effectors bulkier than one aromatic ring (such as biphenyl) or bearing an entirely different distribution of electronegative groups (e. ...200111679066
transposition of deh, a broad-host-range transposon flanked by isppu12, in pseudomonas putida is associated with genomic rearrangements and dehalogenase gene silencing.pseudomonas putida strain pp3 produces two hydrolytic dehalogenases encoded by dehi and dehii, which are members of different deh gene families. the 9.74-kb deh transposon containing dehi and its cognate regulatory gene, dehr(i), was isolated from strain pp3 by using the tol plasmid pww0. deh was fully sequenced and shown to have a composite transposon structure, within which dehi and dehr(i) were divergently transcribed and were flanked on either side by 3.73-kb identical direct repeats. the fl ...200212426347
toluene-degrading antarctic pseudomonas strains from fuel-contaminated soil.two psychrotolerant toluene-degrading pseudomonas spp. were isolated from jp8 jet-fuel-contaminated soils, scott base, antarctica. isolates metabolized meta-toluate as sole carbon source at temperatures ranging from 6 to 30 degrees c. large plasmids (>64kb) were isolated from both isolates. sequence analysis of pcr products amplified using xylb (the gene encoding benzyl alcohol dehydrogenase) primers revealed that isolates 7/167 and 8/46 were 100% and 92% homologous, respectively, to the xylb ge ...200314630048
conjugal tol transfer from pseudomonas putida to pseudomonas aeruginosa: effects of restriction proficiency, toxicant exposure, cell density ratios, and conjugation detection method on observed transfer efficiencies.the effects of restriction proficiency and premating exposure to toxicants on conjugal transfer of the tol plasmid between pseudomonas spp. was investigated by examinations of filter matings. a pseudomonas putida kt2442-derived strain carrying a gfp-tagged variant of the tol plasmid was used as a donor, and both restriction-deficient (pao1162n) and -proficient (pao2002n) pseudomonas aeruginosa strains were used as recipients. the in situ enumeration of conjugation events allowed us to obtain fre ...200515640169
the m-xylene biodegradation capacity of pseudomonas putida mt-2 is submitted to adaptation to abiotic stresses: evidence from expression profiling of xyl genes.the effect of archetypal environmental stresses on expression of the catabolic xyl genes of the tol plasmid pww0 of the m-xylene degrading strain pseudomonas putida mt-2 has been investigated. to this end, a subgenomic dna chip was employed which included structural and regulatory dna sequences of the tol pathway along with selected descriptors of specific physiological conditions. cells were separately exposed to m-xylene under various oxygen tensions, temperatures and nitrogen sources as well ...200616584471
complete sequence determination combined with analysis of transposition/site-specific recombination events to explain genetic organization of incp-7 tol plasmid pww53 and related mobile genetic elements.recent studies have indicated that the evolutionarily common catabolic gene clusters are loaded on structurally diverse toluene-catabolic (tol) plasmids and their residing transposons. to elucidate the mechanisms supporting the diversification of catabolic plasmids and transposons, we determined here the complete 107,929 bp sequence of pww53, a tol plasmid from pseudomonas putida mt53. pww53 was found to belong to the incp-7 incompatibility group that play important roles in the catabolism of se ...200717408691
toluene biodegradation by pseudomonas putida f1: targeting culture stability in long-term operation.the stability of pseudomonas putida f1, a strain harbouring the genes responsible for toluene degradation in the chromosome was evaluated in a bioscrubber under high toluene loadings and nitrogen limiting conditions at two dilution rates (0.11 and 0.27 h(-1)). each experiment was run for 30 days, period long enough for microbial instability to occur considering previously reported studies carried out with bacterial strains encoding the catabolic genes in the tol plasmid. at all tested conditions ...200817487552
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 ...199418615528
transcriptional wiring of the tol plasmid regulatory network to its host involves the submission of the sigma54-promoter pu to the response regulator ppra.implantation of the regulatory circuit of the degradation pathway of tol plasmid pww0 in the native transcriptional network of the host pseudomonas putida involves interplay between plasmid- and chromosome-encoded factors. we have employed a reverse genetics approach to investigate such a molecular wiring by identifying host proteins that form stable complexes with pu, the sigma(54)-dependent promoter of the upper tol operon of pww0. this approach revealed that the pu upstream activating sequenc ...200819138193
The logic layout of the TOL network of Pseudomonas putida pWW0 plasmid stems from a metabolic amplifier motif (MAM) that optimizes biodegradation of m-xylene.ABSTRACT: BACKGROUND: The genetic network of the TOL plasmid pWW0 of the soil bacterium Pseudomonas putida mt-2 for catabolism of m-xylene is an archetypal model for environmental biodegradation of aromatic pollutants. Although nearly every metabolic and transcriptional component of this regulatory system is known to an extraordinary molecular detail, the complexity of its architecture is still perplexing. To gain an insight into the inner layout of this network a logic model of the TOL system ...201122078029
construction and characterization of escherichia coli whole-cell biosensors for toluene and related compounds.the xylr regulatory protein is a transcriptional activator from the tol plasmid of pseudomonas putida mt-2 that is involved in the toluene and benzene degradation pathway. here we describe the construction and laboratory characterization of recombinant biosensors (pglpx plasmids) based on xylr and its cognate promoter (pu). in the pglpx plasmid, the reporter luc gene is under the control of the pu promoter. we evaluated the ability of two distinct nucleotide sequences to function as sd elements ...201020872219
complete nucleotide sequence of tol plasmid pdk1 provides evidence for evolutionary history of incp-7 catabolic plasmids.to understand the mechanisms for structural diversification of pseudomonas-derived toluene-catabolic (tol) plasmids, the complete sequence of a self-transmissible plasmid pdk1 with a size of 128,921 bp from pseudomonas putida hs1 was determined. comparative analysis revealed that (i) pdk1 consisted of a 75.6-kb incp-7 plasmid backbone and 53.2-kb accessory gene segments that were bounded by transposon-associated regions, (ii) the genes for conjugative transfer of pdk1 were highly similar to thos ...201020581207
the regulatory logic of m-xylene biodegradation by pseudomonas putida mt-2 exposed by dynamic modelling of the principal node ps/pr of the tol plasmid.the structure of the extant transcriptional control network of the tol plasmid pww0 born by pseudomonas putida mt-2 for biodegradation of m-xylene is far more complex than one would consider necessary from a mere engineering point of view. in order to penetrate the underlying logic of such a network, which controls a major environmental cleanup bioprocess, we have developed a dynamic model of the key regulatory node formed by the ps/pr promoters of pww0, where the clustering of control elements ...201020553551
a new extant respirometric assay to estimate intrinsic growth parameters applied to study plasmid metabolic burden.start-up phenomena in microbial biokinetic assays are not captured by the most commonly used growth-related equations. in this study we propose a new respirometric experimental design to estimate intrinsic growth parameters that allow us to avoid these limitations without data omission, separate mathematical treatment, or wake-up pulses prior to the analysis. identifiability and sensitivity analysis were performed to confirm the robustness of the new approach for obtaining unique and accurate es ...201019718700
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