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the xyls/pm regulator/promoter system and its use in fundamental studies of bacterial gene expression, recombinant protein production and metabolic engineering.the xyls/pm regulator/promoter system originating from the pseudomonas putida tol plasmid pww0 is widely used for regulated low- and high-level recombinant expression of genes and gene clusters in escherichia coli and other bacteria. induction of this system can be graded by using different cheap benzoic acid derivatives, which enter cells by passive diffusion, operate in a dose-dependent manner and are typically not metabolized by the host cells. combinatorial mutagenesis and selection using th ...201728276630
the impact of succinate trace on pww0 and ortho-cleavage pathway transcription in pseudomonas putida mt-2 during toluene biodegradation.toluene is a pollutant catabolised through the interconnected pww0 (tol) and ortho-cleavage pathways of pseudomonas putida mt-2, while upon succinate and toluene mixtures introduction in batch cultures grown on m9 medium, succinate was previously reported as non-repressing. the effect of a 40 times lower succinate concentration, as compared to literature values, was explored through systematic real-time qpcr monitoring of transcriptional kinetics of the key tol pu, pm and ortho-cleavage pbenr, p ...201728347959
pyridine nucleotide transhydrogenases enable redox balance of pseudomonas putida during biodegradation of aromatic compounds.the metabolic versatility of the soil bacterium pseudomonas putida is reflected by its ability to execute strong redox reactions (e.g., mono- and di-oxygenations) on aromatic substrates. biodegradation of aromatics occurs via the pathway encoded in the archetypal tol plasmid pww0, yet the effect of running such oxidative route on redox balance against the background metabolism of p. putida remains unexplored. to answer this question, the activity of pyridine nucleotide transhydrogenases (that ca ...201627348295
transcriptional kinetics of the cross-talk between the ortho-cleavage and tol pathways of toluene biodegradation in pseudomonas putida mt-2.the tol plasmid promoters are activated by toluene leading to gene expression responsible for the degradation of the environmental signal. benzoate is formed as an intermediate, activating the benr protein of the chromosomal ortho-cleavage pathway that up-regulates the chromosomal pbena promoter and the tol pm promoter resulting in cross-talk between the two networks. herein, the transcriptional kinetics of the pbenr and pbena promoters in conjunction with tol promoters was monitored by real-tim ...201627046069
high-resolution analysis of the m-xylene/toluene biodegradation subtranscriptome of pseudomonas putida mt-2.pseudomonas putida mt-2 metabolizes m-xylene and other aromatic compounds through the enzymes encoded by the xyl operons of the tol plasmid pww0 along with other chromosomally encoded activities. tiling arrays of densely overlapping oligonucleotides were designed to cover every gene involved in this process, allowing dissection of operon structures and exposing the interplay of plasmid and chromosomal functions. all xyl sequences were transcribed in response to aromatic substrates and the 3'-ter ...201626373670
the rna chaperone hfq enables the environmental stress tolerance super-phenotype of pseudomonas putida.the natural physiological regime of the soil bacterium pseudomonas putida involves incessant exposure to endogenous metabolic conflicts and environmental physicochemical insults. yet, the role of assisted small rna-mrna pairing in the stress tolerance super-phenotype that is the trademark of this bacterium has not been accredited. we have thoroughly explored the physiological consequences -in particular those related to exogenous stress - of deleting the hfq gene of p. putida, which encodes the ...201626373442
widening functional boundaries of the σ(54) promoter pu of pseudomonas putida by defeating extant physiological constraints.the extant layout of the σ(54) promoter pu, harboured by the catabolic tol plasmid, pww0, of pseudomonas putida is one of the most complex instances of endogenous and exogenous signal integration known in the prokaryotic domain. in this regulatory system, all signal inputs are eventually translated into occupation of the promoter sequence by either of two necessary components: the m-xylene responsive transcriptional factor xylr and the σ(54) containing form of rna polymerase. modelling of these ...201525560994
freeing pseudomonas putida kt2440 of its proviral load strengthens endurance to environmental stresses.2.6% of the genome of the soil bacterium pseudomonas putida kt2440 encodes phage-related functions, but the burden of such opportunistic dna on the host physiology is unknown. each of the four apparently complete prophages borne by this strain was tested for stability, spontaneous excision and ability to cause lysis under various stressing conditions. while prophages p3 (pp2266-pp2297) and p4 (pp1532-1584) were discharged from the genome at a detectable rate, their induction failed otherwise to ...201524762028
chemical reactivity drives spatiotemporal organisation of bacterial metabolism.in this review, we examine how bacterial metabolism is shaped by chemical constraints acting on the material and dynamic layout of enzymatic networks and beyond. these are moulded not only for optimisation of given metabolic objectives (e.g. synthesis of a particular amino acid or nucleotide) but also for curbing the detrimental reactivity of chemical intermediates. besides substrate channelling, toxicity is avoided by barriers to free diffusion (i.e. compartments) that separate otherwise incomp ...201525227915
a second chromosomal copy of the cata gene endows pseudomonas putida mt-2 with an enzymatic safety valve for excess of catechol.pseudomonas putida mt-2 harbours two different routes for catabolism of catechol, namely one meta pathway encoded by the xyl genes of the tol plasmid pww0 and one ortho pathway determined by the chromosomal ben and cat genes. p. putida mt-2 has a second chromosomal copy of the cata gene (named cata2) located downstream of the ben operon that encodes an additional catechol-1,2-dioxygenase. the metabolic and regulatory phenotypes of strains lacking one enzyme, the other and both of them in cells w ...201424341396
the pww0 plasmid imposes a stochastic expression regime to the chromosomal ortho pathway for benzoate metabolism in pseudomonas putida.environmental plasmids often expand the metabolic repertoire of bacteria that carry them, but they also interfere with the biochemical and genetic network of the host. the pww0 plasmid born by pseudomonas putida mt-2 encodes the tol pathway for degradation of toluene/m-xylene through production of intermediate compounds benzoate/3-methylbenzoate. these can be also recognized as substrates by the chromosomally encoded ben and cat gene products, thereby creating a manifest regulatory and biochemic ...201425848660
deepening tol and tou catabolic pathways of pseudomonas sp. ox1: cloning, sequencing and characterization of the lower pathways.pseudomonas sp. ox1 is able to metabolize toluene and o-xylene through the tou catabolic pathway, whereas its mutant m1 strain was found to be able to use m- and p-xylene as carbon and energy source, using the tol catabolic pathway. here we report the complete nucleotide sequence of the phe lower operon of the tou catabolic pathway, and the sequence of the last four genes of the xyl-like lower operon of the tol catabolic pathway. dna sequence analysis shows the gene order within the operons to b ...201323009925
accumulation of inorganic polyphosphate enables stress endurance and catalytic vigour in pseudomonas putida kt2440.accumulation of inorganic polyphosphate (polyp), a persistent trait throughout the whole tree of life, is claimed to play a fundamental role in enduring environmental insults in a large variety of microorganisms. the share of polyp in the tolerance of the soil bacterium pseudomonas putida kt2440 to a suite of physicochemical stresses has been studied on the background of its capacity as a host of oxidative biotransformations.201323687963
complete nucleotide sequence of the self-transmissible tol plasmid pd2rt provides new insight into arrangement of toluene catabolic plasmids.in the present study we report the complete nucleotide sequence of the toluene catabolic plasmid pd2rt of pseudomonas migulae strain d2rt isolated from baltic sea water. the pd2rt is 129,894 base pairs in size with an average g+c content of 53.75%. a total of 135 open reading frames (orfs) were predicted to encode proteins, among them genes for catabolism of toluene, plasmid replication, maintenance and conjugative transfer. orfs encoding proteins with putative functions in stress response, tran ...201324095800
a spatiotemporal view of plasmid loss in biofilms and planktonic cultures.this commentary by madsen, burmølle, and sørensen discusses the article non-invasive in situ monitoring and quantification of tol plasmid segregational loss within pseudomonas putida biofilms by ma, katzenmeyer, and bryers. (2013. biotechnol bioeng. 110(11):2949-2958. doi: 10.1002/bit.24953).201324014288
non-invasive in situ monitoring and quantification of tol plasmid segregational loss within pseudomonas putida biofilms.methods for the detection of plasmid loss in natural environments have typically relied on replica plating, selective markers and pcr. however, these traditional methods have the limitations of low sensitivity, underestimation of specific cell populations, and lack of insightful data for non-homogeneous environments. we have developed a non-invasive microscopic analytical method to quantify local plasmid segregational loss from a bacterial population within a developing biofilm. the probability ...201323633286
functionality of the tol plasmid under varying environmental conditions following conjugal transfer.conjugation of catabolic plasmids in contaminated environments is a naturally occurring horizontal gene transfer phenomenon, which could be utilized in genetic bioaugmentation. the potentially important parameters for genetic bioaugmentation include gene regulation of transferred catabolic plasmids that may be controlled by the genetic characteristics of transconjugants as well as environmental conditions that may alter the expression of the contaminant-degrading phenotype. this study showed tha ...201322367613
the three-species consortium of genetically improved strains cupriavidus necator rw112, burkholderia xenovorans rw118, and pseudomonas pseudoalcaligenes rw120 grows with technical polychlorobiphenyl, aroclor 1242.burkholderia xenovorans lb400, cupriavidus necator h850, and pseudomonas pseudoalcaligenes kf707 are bacterial strains able to mineralize biphenyl and to co-oxidize many of its halogenated derivatives (pcbs). only strain lb400 also mineralizes a few mono- and dichlorobiphenyls, due to the presence of a functioning chlorocatechol pathway. here, we used a tn5-based minitransposon shuttle system to chromosomically introduce genes tcbrcdef, encoding the chlorocatechol pathway into kf707, and genes c ...201323658554
genetic bioaugmentation as an effective method for in situ bioremediation: functionality of catabolic plasmids following conjugal transfers.genetic bioaugmentation is an in situ bioremediation method that stimulates horizontal transfer of catabolic plasmids between exogenous donor cells and indigenous bacteria to increase the biodegradation potential of contaminants. a critical outcome of genetic bioaugmentation is the expression of an active catabolic phenotype upon plasmid conjugation. using a pww0-derivative tol plasmid, we showed that certain genetic characteristics of the recipient bacteria, including genomic guanine-cytosine ( ...201222705839
increasing signal specificity of the tol network of pseudomonas putida mt-2 by rewiring the connectivity of the master regulator xylr.prokaryotic transcription factors (tfs) that bind small xenobiotic molecules (e.g., tfs that drive genes that respond to environmental pollutants) often display a promiscuous effector profile for analogs of the bona fide chemical signals. xylr, the master tf for expression of the m-xylene biodegradation operons encoded in the tol plasmid pww0 of pseudomonas putida, responds not only to the aromatic compound but also, albeit to a lesser extent, to many other aromatic compounds, such as 3-methylbe ...201223071444
impacts of organic carbon availability and recipient bacteria characteristics on the potential for tol plasmid genetic bioaugmentation in soil slurries.the effectiveness of genetic bioaugmentation relies on efficient plasmid transfer between donor and recipient cells as well as the plasmid's phenotype in the recipient cell. in the present study, the effects of varying organic carbon substrates, initial recipient-to-donor cell density ratios, and mixtures of known recipient bacterial strains on the conjugation and function of a tol plasmid were tested in sterile soil slurry batch reactors. the presence of soil organic carbon was sufficient in en ...201222743182
stochasticity of tol plasmid catabolic promoters sets a bimodal expression regime in pseudomonas putida mt-2 exposed to m-xylene.the expression dynamics of the catabolic promoters of the tol plasmid pww0 has been examined in single cells of pseudomonas putida mt-2 exposed to m-xylene. to this end, we employed an á la carte bi-cistronic gfp-lacz reporter system for generating monocopy transcriptional fusions to each of the four promoters (pr, ps, pu and pm) of the regulatory network. whereas expression of xylr (the master regulatory gene of the tol system) behaved in a unimodal fashion, the activation of pu and pm displaye ...201222845424
cooperative amino acid changes shift the response of the σ⁵⁴-dependent regulator xylr from natural m-xylene towards xenobiotic 2,4-dinitrotoluene.xylr is a σ⁵⁴-dependent transcriptional factor of pseudomonas putida that activates the pu promoter of the tol plasmid upon binding its natural effector, m-xylene. the search for mutants of the signal-sensing module of xylr that respond to the xenobiotic compound 2,4-dinitrotoluene recurrently yields protein variants with a broad effector range. these mutants had amino acid changes not only in the effector recognition moiety (a module), but also in the inter-domain b linker of the protein. a ran ...201121205010
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
An efficient design strategy for a whole-cell biosensor based on engineered ribosome binding sequences.In prokaryotes, the ribosome binding sequence (RBS), located in the 5' untranslated region (5' UTR) of an mRNA, plays a critical role in enhancing mRNA translation and stability. To evaluate the effect of the RBS on the sensitivity and signal intensity of an environmental whole-cell biosensor, three Escherichia coli-based biosensors that respond to benzene, toluene, ethylbenzene, and the xylenes (BTEX) were constructed; the three biosensors have the same Pu promoter and xylR regulator from the P ...201121947012
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
effect of carbon source addition on toluene biodegradation by an escherichia coli dh5alpha transconjugant harboring the tol plasmid.horizontal gene transfer (hgt) of plasmids is a naturally occurring phenomenon which could be manipulated for bioremediation applications. specifically, hgt may prove useful to enhance bioremediation through genetic bioaugmentation. however, because the transfer of a plasmid between donor and recipient cells does not always result in useful functional phenotypes, the conditions under which hgt events result in enhanced degradative capabilities must first be elucidated. the objective of this stud ...201020506384
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
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
high stability and fast recovery of expression of the tol plasmid-carried toluene catabolism genes of pseudomonas putida mt-2 under conditions of oxygen limitation and oscillation.pseudomonas putida mt-2 harbors the tol plasmid (pwwo), which contains the genes encoding the enzymes necessary to degrade toluene aerobically. the xyl genes are clustered in the upper operon and encode the enzymes of the upper pathway that degrade toluene to benzoate, while the genes encoding the enzymes of the lower pathway (meta-cleavage pathway) that are necessary for the conversion of benzoate to tricarboxylic acid cycle intermediates, are encoded in a separate operon. in this study, the ef ...201020709833
tol plasmid carriage enhances biofilm formation and increases extracellular dna content in pseudomonas putida kt2440.adherent growth of pseudomonas putida kt2440 with and without the tol plasmid (pwwo) at the solid-liquid and air-liquid interface was examined. we compared biofilm formation on glass in flow cells, and assayed pellicle (air-liquid interface biofilm) formation in stagnant liquid cultures by confocal laser scanning microscopy. the tol-carrying strains formed pellicles and thick biofilms, whereas the same strains without the plasmid displayed little adherent growth. microscopy using fluorescent nuc ...201020846143
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
an individual-based approach to explain plasmid invasion in bacterial populations.we present an individual-based experimental framework to identify and estimate the main parameters governing bacterial conjugation at the individual cell scale. from this analysis, we have established that transient periods of unregulated plasmid transfer within newly formed transconjugant cells, together with contact mechanics arising from cellular growth and division, are the two main processes determining the emergent inability of the pww0 tol plasmid to fully invade spatially structured pseu ...201021091520
bioaugmentation of microbial communities in laboratory and pilot scale sequencing batch biofilm reactors using the tol plasmid.the aim of this study was to investigate the effectiveness of bioaugmentation and transfer of plasmid pwwo (tol plasmid) to mixed microbial populations in pilot and laboratory scale sequencing batch biofilm reactors (sbbrs) treating synthetic wastewater containing benzyl alcohol (ba) as a model xenobiotic. the plasmid donor was a pseudomonas putida strain chromosomally tagged with the gene for the red fluorescent protein carrying a green fluorescent protein labeled tol plasmid, which confers deg ...200919010662
conditions required for the stimulation of bioluminescence activity of the genetically engineered bacteria, p. putida mt-2 kg1206, preserved by deep-freezing.herein the conditions required for the stimulation of bioluminescence activity in a genetically engineered strain of pseudomonas putida mt-2 kg1206, containing the intact tol plasmid and a constructed plasmid with the p(m)-lux gene, are reported upon. both sodium lactate (sl) and potassium nitrate (kno(3)) were able to stimulate the bioluminescence activity, but a greater increase was observed with nitrogen amendment. this selected stimulant was then tested on reconstituted cells that had been p ...200919176233
competition triggers plasmid-mediated enhancement of substrate utilisation in pseudomonas putida.competition between species plays a central role in the activity and structure of communities. stable co-existence of diverse organisms in communities is thought to be fostered by individual tradeoffs and optimization of competitive strategies along resource gradients. outside the laboratory, microbes exist as multispecies consortia, continuously interacting with one another and the environment. survival and proliferation of a particular species is governed by its competitive fitness. therefore, ...200919557171
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
xyls-pm promoter interactions through two helix-turn-helix motifs: identifying xyls residues important for dna binding and activation.the xyls protein is the positive transcription regulator of the tol plasmid meta-cleavage pathway operon pm. xyls belongs to the arac family of transcriptional regulators and exhibits an n-terminal domain involved in effector recognition, and a c-terminal domain, made up of seven alpha-helices conforming two helix-turn-helix dna-binding domains. alpha-helix 3 and alpha-helix 6 are the recognition helices. in consonance with xyls structural organization, pm exhibits a bipartite dna-binding motif ...200818005985
bioaugmentation of aerobic microbial granules with pseudomonas putida carrying tol plasmid.this paper describes results of a successful bioaugmentation experiment on aerobic granular sludge using pseudomonas putida kt2442 cells bearing the tol (pwwo) plasmid. the methodology was designed to monitor incorporation of the added donor cells into pre-existent microbial granules and the subsequent plasmid transfer to the autochthonous microbial community using shake flask microcosms. expression of reporter proteins (gfp and dsred) allowed in situ monitoring of donor cell attachment and plas ...200818076969
[study on method of acclimation and screening of a highly effective toluene-degrading bacterium].to establish a highly effective toluene-degrading bacterium acclimation and screening method.200818589590
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
the ttgghi solvent efflux pump operon of pseudomonas putida dot-t1e is located on a large self-transmissible plasmid.pseudomonas putida dot-t1e is a solvent-tolerant strain able to grow in the presence of > 1% (v/v) toluene in the culture medium. a set of multidrug efflux pumps have been found to play a major role in the tolerance of this bacterium to organic solvents (rojas et al., j bacteriol 183: 3967-3973). in the course of studies of the mechanisms underlying solvent tolerance in dot-t1e, we isolated a spontaneous solvent-sensitive mutant derivative which had lost the genes encoding the ttgghi efflux pump ...200717504492
simultaneous catabolite repression between glucose and toluene metabolism in pseudomonas putida is channeled through different signaling pathways.pseudomonas putida kt2440(pww0) can use toluene via the tol plasmid-encoded catabolic pathways and can use glucose via a series of three peripheral chromosome-encoded routes that convert glucose into 6-phosphogluconate (6pg), namely, the glucokinase pathway, in which glucose is transformed to 6pg through the action of glucokinase and glucose-6-phosphate dehydrogenase. alternatively, glucose can be oxidized to gluconate, which can be phosphorylated by gluconokinase to 6pg or oxidized to 2-ketoglu ...200717616587
tol plasmid transfer during bacterial conjugation in vitro and rhizoremediation of oil compounds in vivo.molecular profiling methods for horizontal transfer of aromatics-degrading plasmids were developed and applied during rhizoremediation in vivo and conjugations in vitro. pww0 was conjugated from pseudomonas to rhizobium. the xyle gene was detected both in rhizobium galegae bv. officinalis and bv. orientalis, but it was neither stably maintained in orientalis nor functional in officinalis. tol plasmids were a major group of catabolic plasmids among the bacterial strains isolated from the oil-cont ...200717000041
new insights on toluene biodegradation by pseudomonas putida f1: influence of pollutant concentration and excreted metabolites.the influence of toluene concentration on the specific growth rate, cellular yield, specific co(2), and metabolite production by pseudomonas putida f1 (ppf1) was investigated. both cellular yield and specific co(2) production remained constant at 1.0 +/- 0.1 g biomass dry weight (dw) g(-1) toluene and 1.91 +/- 0.31 g co(2) g(-1) biomass, respectively, under the tested range of concentrations (2-250 mg toluene l(-1)). the specific growth rate increased up to 70 mg toluene l(-1). further increases ...200717136537
growth of the genetically engineered strain cupriavidus necator rw112 with chlorobenzoates and technical chlorobiphenyls.cupriavidus necator (formerly ralstonia eutropha) strain h850 is known to grow on biphenyl, and to co-oxidize congeners of polychlorinated biphenyls (pcbs). using a tn5-based minitransposon shuttle system and the tol plasmid, the rational construction of hybrids of h850 was achieved by subsequent introduction of three distinct elements carrying 11 catabolic loci from three other biodegrading bacteria into the parent strain, finally yielding c. necator rw112. the new genetic elements introduced i ...200717185547
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
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
in vivo drafting of single-chain antibodies for regulatory duty on the sigma54-promoter pu of the tol plasmid.the identification of single-chain antibodies (scfvs) that interfere in vivo with the building of the complex that activate the prokaryotic, sigma54-dependent promoter pu of the catabolic tol plasmid pww0 is reported. to this end, a phage m13 library of scfvs was raised against the cognate prokaryotic enhancer-binding activator, xylr. the scfv pool was then expressed intracellularly in a reporter pu-lacz strain of escherichia coli designed to permit formation of intramolecular disulphide bonds i ...200616689797
genetic diversity of culturable bacteria in oil-contaminated rhizosphere of galega orientalis.a collection of 50 indigenous meta-toluate tolerating bacteria isolated from oil-contaminated rhizosphere of galega orientalis on selective medium was characterized and identified by classical and molecular methods. 16s rdna partial sequencing showed the presence of five major lineages of the bacteria domain. gram-positive rhodococcus, bacillus and arthrobacter and gram-negative pseudomonas were the most abundant genera. only one-fifth of the strains that tolerated m-toluate also degraded m-tolu ...200616055251
evolutionary algorithms and flow cytometry to examine the parameters influencing transconjugant formation.an evolutionary algorithm was used to determine the optimal combination of parameters for transconjugant formation. as a model system, a gfp tagged tol plasmid pww0 was chosen to examine transfer from pseudomonas putida to escherichia coli. a comparison of flow cytometry results with plating and microscopy showed that the majority of transconjugants were not culturable. the transconjugant ratio therefore was determined by flow cytometry. the evolutionary algorithm showed that the optimal conditi ...200616420611
the upstream-activating sequences of the sigma54 promoter pu of pseudomonas putida filter transcription readthrough from upstream genes.although the m-xylene-responsive sigma54 promoter pu of pseudomonas putida mt-2, borne by the tol plasmid pwwo, is one of the strongest known promoters in vivo, its base-line level in the absence of its aromatic inducer is below the limit of any detection procedure. this is unusual because regulatory networks (such as the one to which pu belongs) can hardly escape the noise caused by intrinsic fluctuations in background transcription, including that transmitted from upstream promoters. this stud ...200616510445
integration of signals through crc and ptsn in catabolite repression of pseudomonas putida tol plasmid pww0.toluene degradation in pseudomonas putida kt2440 pww0 plasmid is subjected to catabolite repression. pu and p(s1) promoters of the pww0 tol plasmid are down-regulated in vivo during exponential growth in rich medium. in cells growing on minimal medium, yeast extract (ye) addition mimics exponential-phase rich medium repression of these promoters. we have constructed and tested mutants in a series of global regulators described in pseudomonas. we describe that a mutant in crc (catabolite repressi ...200516085802
inferring the genetic network of m-xylene metabolism through expression profiling of the xyl genes of pseudomonas putida mt-2.a subgenomic array of structural and regulatory genes of the tol plasmid pww0 of pseudomonas putida mt-2 has been constructed to sort out the interplay between m-xylene catabolism and the environmental stress brought about by this aromatic chemical. to this end, xyl sequences were spotted along with groups of selected p. putida genes, the transcription of which become descriptors of distinct physiological conditions. the expression of the tol pathway in response to pathway substrates was thus pr ...200516135224
transcriptional activation of quinoline degradation operons of pseudomonas putida 86 by the arac/xyls-type regulator oxos and cross-regulation of the pqorm promoter by xyls.the quinoline-degradative gene cluster (oxoo, open reading frames 1 to 6 [orf1 to -6], qormsl, orf7 to -9, oxor) of pseudomonas putida 86 consists of several overlapping operons controlled in response to quinoline by the master promoter poxoo and internal promoters porf3, pqorm, and poxor. orf7 to -9, presumed to be important for maturation of the molybdenum hydroxylase quinoline 2-oxidoreductase, are also weakly transcribed independently of quinoline. expression of the oxos gene, located upstre ...200516332855
m-xylene-responsive pu-pnifh hybrid sigma54 promoters that overcome physiological control in pseudomonas putida kt2442.the sequences surrounding the -12/-24 motif of the m-xylene-responsive sigma54 promoter pu of the pseudomonas putida tol plasmid pww0 were replaced by various dna segments of the same size recruited from pnifh sigma54 promoter variants known to have various degrees of efficacy and affinity for sigma54-rna polymerase (rnap). in order to have an accurate comparison of the output in vivo of each of the hybrids, the resulting promoters were recombined at the same location of the chromosome of p. put ...200515601696
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
a dna polymerase v homologue encoded by tol plasmid pww0 confers evolutionary fitness on pseudomonas putida under conditions of environmental stress.plasmids in conjunction with other mobile elements such as transposons are major players in the genetic adaptation of bacteria in response to changes in environment. here we show that a large catabolic tol plasmid, pww0, from pseudomonas putida carries genes (rulab genes) encoding an error-prone dna polymerase pol v homologue which increase the survival of bacteria under conditions of accumulation of dna damage. a study of population dynamics in stationary phase revealed that the presence of pww ...200516030214
novel physiological modulation of the pu promoter of tol plasmid: negative regulatory role of the tura protein of pseudomonas putida in the response to suboptimal growth temperatures.from crude protein extracts of pseudomonas putida kt2440, we identified a small protein, tura, able to bind to dna fragments bearing the entire pu promoter sequence of the tol plasmid. the knock-out inactivation of the tura gene resulted in enhanced transcription initiation from the pu promoter, initially suggesting a negative regulatory role of tura on pu expression. ectopic expression of tura both in p. putida and in escherichia coli reporter strains and transcription in vitro of the pu promot ...200414672954
recombinant escherichia coli for the biomonitoring of benzene and its derivatives in the air.for protection against environmental deterioration, pollutants should be reduced as much as possible. therefore, a sensitive detection method for air pollutants is required, particularly for benzene, a compound with mutagenic, teratogenic, and carcinogenic properties. some microorganisms have a number of enzymes that degrade organic compounds. the genetic information for many of these enzymes is codified in plasmids that usually comprise some elements such as proteins and rna for their replicati ...200414759656
genetic evidence that catabolites of the entner-doudoroff pathway signal c source repression of the sigma54 pu promoter of pseudomonas putida.glucose and other c sources exert an atypical form of catabolic repression on the sigma54-dependent promoter pu, which drives transcription of an operon for m-xylene degradation encoded by the tol plasmid pww0 in pseudomonas putida. we have used a genetic approach to identify the catabolite(s) shared by all known repressive c sources that appears to act as the intracellular signal that triggers downregulation of pu. to this end, we reconstructed from genomic data the pathways for metabolism of r ...200415576775
combined use of different gfp reporters for monitoring single-cell activities of a genetically modified pcb degrader in the rhizosphere of alfalfa.single-cell localization and activity of pseudomonas fluorescens f113, colonizing alfalfa roots, were monitored using fusions of the escherichia coli rrnbp1 ribosomal promoter and gfp genes encoding green fluorescent protein (gfp) of different stability. the monitoring systems permitted non-destructive in situ detection of f113rifpcb cells on the entire root system grown in both the presence and absence of 3-chlorobiphenyl (pcb-2). the root tip and sites of lateral root emergence were found to b ...200419712397
complete nucleotide sequence of carbazole/dioxin-degrading plasmid pcar1 in pseudomonas resinovorans strain ca10 indicates its mosaicity and the presence of large catabolic transposon tn4676.the car and ant operons originally isolated from pseudomonas resinovorans strain ca10 contain the genes encoding the carbazole/dioxin-degrading enzymes and anthranilate 1,2-dioxygenase, respectively, and are located on the plasmid pcar1. the entire nucleotide sequence of pcar1 was determined to elucidate the mechanism by which the car operon may have been assembled and distributed in nature. pcar1 is a 199,035-bp circular plasmid, and carries 190 open reading frames. although the incompatibility ...200312547188
sigma 54 levels and physiological control of the pseudomonas putida pu promoter.the cellular levels of the alternative sigma factor sigma(54) of pseudomonas putida have been examined in a variety of growth stages and culture conditions with a single-chain fv antibody tailored for detection of scarce proteins. the levels of sigma(54) were also monitored in p. putida strains with knockout mutations in ptso or ptsn, known to be required for the c-source control of the sigma(54)-dependent pu promoter of the tol plasmid. our results show that approximately 80 +/- 26 molecules of ...200312754236
identification and characterization of the conjugal transfer region of the pcg1 plasmid from naphthalene-degrading pseudomonas putida cg1.hybridization and restriction fragment length polymorphism data (k. g. stuart-keil, a. m. hohnstock, k. p. drees, j. b. herrick, and e. l. madsen, appl. environ. microbiol. 64:3633-3640, 1998) have shown that pcg1, a naphthalene catabolic plasmid carried by pseudomonas putida cg1, is homologous to the archetypal naphthalene catabolic plasmid, pdtg1, in p. putida ncib 9816-4. sequencing of the latter plasmid allowed pcr primers to be designed for amplifying and sequencing the conjugal transfer re ...200312788725
dual labeling of pseudomonas putida with fluorescent proteins for in situ monitoring of conjugal transfer of the tol plasmid.we describe here a dual-labeling technique involving the green fluorescent protein (gfp) and the red fluorescent protein (dsred) for in situ monitoring of horizontal gene transfer via conjugation. a gfpmut3b-tagged derivative of narrow-host-range tol plasmid (pwwo) was delivered to pseudomonas putida kt2442, which was chromosomally labeled with dsred by transposon insertion via biparental mating. green and red fluorescent proteins were coexpressed in donor p. putida cells. cells expressing both ...200312902279
design of catabolic cassettes for styrene biodegradation.a broad-host range metabolic cassette has been designed that, under the control of the ptac promoter, expresses the sytabcd catabolic genes from pseudomonas sp. y2, which are responsible for the transformation of styrene into phenylacetic acid (styrene upper pathway). this novel cassette confers to phenylacetic acid-degrading bacteria the ability to grow efficiently on styrene as the sole carbon and energy source. by combining both the sty cassette and the archetypal pww0 tol plasmid into the we ...200312906358
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
direct atomic force microscopy visualization of integration host factor-induced dna bending structure of the promoter regulatory region on the pseudomonas tol plasmid.atomic force microscopy (afm) was used to analyze dna bending induced by integration host factor (ihf). the direct afm visualization of ihf-dna complexes on the op1 promoter regulatory regions on the pseudomonas tol plasmid showed that there was no intrinsic dna bend in the op1 promoter region, but a sharp dna bend was induced by binding of ihf to the region between the upstream regulatory sequence and the promoter sequence. the dna bending angles were distributed with a mean bend angle of 123 d ...200211846413
xyls activator and rna polymerase binding sites at the pm promoter overlap.transcription from the tol plasmid meta-cleavage pathway operon, pm, depends on the xyls protein being activated by a benzoate effector. the xyls binding sites are two imperfect 5'-tgcan(6)ggnta-3' direct repeats located between positions -70/-56 and -49/-35 [gonzález-pérez et al. (1999) j. biol. chem. 274, 2286-2290]. an intrinsic bending of 40 degrees, which is not essential for transcription, is centered at position -43. we have determined the potential overlap between the xyls and rna polyme ...200212023029
chloroplast-type ferredoxin involved in reactivation of catechol 2,3-dioxygenase from pseudomonas sp. s 47.pseudomonas sp. s-47 is capable of degrading catechol and 4-chlorocatechol via the meta-cleavage pathway. xylte products catalyze the dioxygenation of the aromatics. the xylt of the strain s-47 is located just upstream of the xyle gene. xylt is a typical chloroplast-type ferredoxin, which is characterized by 4 cystein residues that are located at positions 41, 46, 49, and 81. the chloroplast-type ferredoxin of pseudomonas sp. s-47 exhibited a 98% identity with that of p. putida mt-2 (tol plasmid ...200212297005
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
complete sequence of the incp-9 tol plasmid pww0 from pseudomonas putida.the tol plasmid pww0 (117 kb) is the best studied catabolic plasmid and the archetype of the incp-9 plasmid incompatibility group from pseudomonas. it carries the degradative (xyl) genes for toluenes and xylenes within catabolic transposons tn4651 and tn4653. analysis of the complete pww0 nucleotide sequence revealed 148 putative open reading frames. of these, 77 showed similarity to published sequences in the available databases predicting functions for: plasmid replication, stable maintenance ...200212534468
the chlorobenzoate dioxygenase genes of burkholderia sp. strain nk8 involved in the catabolism of chlorobenzoates.burkholderia sp. nk8 grows abundantly on 3-chlorobenzoate (3cb),4-chlorobenzoate (4cb) and benzoate. the genes encoding the oxidation of (chloro)benzoates (cbeabcd) and catechol (cata, catbc), the lysr-type regulatory gene cber and the gene cbee with unknown function, all of which form a single cluster in nk8, were cloned and analysed. the protein sequence of chlorobenzoate 1,2-dioxygenase (cbeabc) is 50-65% identical to the benzoate dioxygenase (benabc) of acinetobacter sp. adp1, toluate dioxyg ...200111160806
rational design of a bacterial transcriptional cascade for amplifying gene expression capacity.cascade regulatory circuits have been described that control numerous cell processes, and may provide models for the design of artificial circuits with novel properties. here we describe the design of a transcriptional regulatory cascade to amplify the cell response to a given signal. we used the salicylate-responsive activators of pseudomonas putida nahr of the naphthalene degradation plasmid nah7 and xyls2, a mutant regulator of the tol plasmid for catabolism of m-xylene and their respective c ...200111160899
metabolic engineering of bacteria for environmental applications: construction of pseudomonas strains for biodegradation of 2-chlorotoluene.in this article, we illustrate the challenges and bottlenecks in the metabolic engineering of bacteria destined for environmental bioremediation, by reporting current efforts to construct pseudomonas strains genetically designed for degradation of the recalcitrant compound 2-chlorotoluene. the assembled pathway includes one catabolic segment encoding the toluene dioxygenase of the tod system of pseudomonas putida f1 (todc1c2ba), which affords the bioconversion of 2-chlorotoluene into 2-chloroben ...200111165359
expression of 2-halobenzoate dioxygenase genes (cbdsabc) involved in the degradation of benzoate and 2-halobenzoate in burkholderia sp. th2.burkholderia sp. th2, isolated from soil, utilizes 2-chlorobenzoate (2cb) and benzoate (ba) as its sole source of carbon and energy. the genes for 2-halobenzoate dioxygenase (cbdabc) from burkholderia sp. th2 were cloned and sequenced. the predicted amino acid sequences of all the gene products are highly similar to the cbd gene products of pseudomonas sp. 2cbs. disruption of the promoter region of cbda resulted in loss of growth on 2cb and ba, indicating that these genes are involved in the gro ...200111179677
dual system to reinforce biological containment of recombinant bacteria designed for rhizoremediation.active biological containment (abc) systems have been designed to control at will the survival or death of a bacterial population. these systems are based on the use of a killing gene, e.g., a porin-inducing protein such as the one encoded by the escherichia coli gef gene, and a regulatory circuit that controls expression of the killing gene in response to the presence or absence of environmental signals. an abc system for recombinant microorganisms that degrade a model pollutant was designed on ...200111375176
toluene mineralization and growth potential of pseudomonas putida paw164 under toluene-limiting conditions.toluene-induced cells of pseudomonas putida paw164 (pwwo-164) were monitored for growth potential, maintaining the tol plasmid, and potential toluene mineralization activity in toluene-amended and nonamended soil. a follow-up study was done in a carbon-free mineral salts solution to obtain further information on physiological changes that occur during starvation. these studies showed that there was a larger decline in colony forming units (cfus) recovered on a toluate- or benzoate-defined minera ...200111462134
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
identification and characterization of tn4656, a novel class ii transposon carrying a set of toluene-degrading genes from tol plasmid pww53.it has been reported that the toluene-degrading (xyl) genes from pseudomonas putida plasmid pww53 are able to translocate to broad-host-range drug resistance plasmid rp4, and pww53-4 is one of the smallest rp4 derivatives (h. keil, s. keil, r. w. pickup, and p. a. williams, j. bacteriol. 164:887-895, 1985). our investigation of pww53-4 in this study demonstrated that such a translocated region that is 39 kb long is a transposon. this mobile element, tn4656, was classified as a class ii transposo ...200111591664
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
retrotransfer of dna in the rhizosphere.retrotransfer of dna refers to the phenomenon by which a plasmid travels from a host strain to a recipient one and returns to the original host, bringing with it dna from the recipient. the resultant host strain with dna from the recipient is called a retrotransconjugant. the retrotransfer phenomenon mediated by the tol plasmid pww0 and other plasmids has been documented on plates under optimal laboratory culture conditions, but never under natural conditions. in this work, we show that retrotra ...200011200433
effect of inoculation of a tol plasmid containing mycorrhizosphere bacterium on development of scots pine seedlings, their mycorrhizosphere and the microbial flora in m-toluate-amended soil.the purpose of this study was to evaluate the influence of introduced bacteria containing a contaminant degrading plasmid on the growth and survival of pine seedlings and mycorrhizosphere microbial flora in contaminated soil. the pseudomonas fluorescens strain os81, originally isolated from fungal hyphae in contaminated soil, was supplied with the tol plasmid pww0::km (to generate os81(pww0::km)) and inoculated in humus-soil microcosms with and without pine seedlings mycorrhized with suillus bov ...200010640666
genetic evidence of distinct physiological regulation mechanisms in the sigma(54) pu promoter of pseudomonas putida.the activity of the toluene-responsive sigma(54) pu promoter of the pww0 tol plasmid of pseudomonas putida is down-regulated in vivo during exponential growth in rich medium and also by the presence of glucose in the culture. although the pu promoter already performs poorly during log growth in minimal medium when amended with casamino acids, the addition of glucose further decreased by two- to threefold the accumulation of beta-galactosidase in a pu-lacz reporter p. putida strain. since pu was ...200010648520
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
cloning and expression of ntnd, encoding a novel nad(p)(+)-independent 4-nitrobenzyl alcohol dehydrogenase from pseudomonas sp. strain tw3.pseudomonas sp. strain tw3 is able to metabolize 4-nitrotoluene to 4-nitrobenzoate and toluene to benzoate aerobically via a route analogous to the upper pathway of the tol plasmids. we report the cloning and characterization of a benzyl alcohol dehydrogenase gene (ntnd) which encodes the enzyme for the catabolism of 4-nitrobenzyl alcohol and benzyl alcohol to 4-nitrobenzaldehyde and benzaldehyde, respectively. the gene is located downstream of the previously reported ntn gene cluster. ntnd bear ...200010809692
physiological and genetic comparison of two aromatic hydrocarbon-degrading sphingomonas strains.sphingomonas yanoikuyae strain b1 is able to degrade a wider range of aromatic hydrocarbons than s. paucimobilis strain tne12 can degrade. various culture techniques were used to corroborate that b1 used m-xylene, biphenyl, toluene, naphthalene, and phenanthrene as sole carbon and energy sources. in contrast, tne12 could not mineralize m-xylene, biphenyl, toluene, or naphthalene. however, fluoranthene served as carbon and energy source for tne12 but not b1. southern blots were performed using th ...200010850662
mutational analysis of the highly conserved c-terminal residues of the xyls protein, a member of the arac family of transcriptional regulators.the xyls protein of the tol plasmid of pseudomonas putida belongs to the so-called arac/xyls family of regulators, that includes more than 100 different bacterial proteins. a conserved stretch of about 100 amino acids is present at the c-terminal end. this conserved region is believed to contain seven alpha-helices, including two helix-turn-helix (hth) dna binding motifs (alpha(2)-t-alpha(3) and alpha(5)-talpha-(6)), connected by a linker alpha-helix (alpha(4)), and two flanking alpha-helices (a ...200010913634
in vivo and in vitro effects of (p)ppgpp on the sigma(54) promoter pu of the tol plasmid of pseudomonas putida.the connection between the physiological control of the sigma(54)-dependent pu promoter of the tol plasmid pww0 of pseudomonas putida and the stringent response mediated by the alarmone (p)ppgpp has been examined in vivo an in vitro. to this end, the key regulatory elements of the system were faithfully reproduced in an escherichia coli strain and assayed as lacz fusions in various genetic backgrounds lacking (p)ppgpp or overexpressing rela. neither the responsiveness of pu to 3-methyl benzylalc ...200010940009
visualization of dna-protein intermediates during activation of the pu promoter of the tol plasmid of pseudomonas putida.the atp-dependent multimerization process undergone by the sigma(54)-dependent activator xylr of the tol plasmid pww0 of pseudomonas putida when bound to the upstream activating sequences (uas) of the cognate pu: promoter was examined by transmission electron microscopy (tem). to this end, supercoiled dna templates were combined with increasing concentrations of the constitutive xylr variant xylrdeltaa, with or without atp or its non-hydrolysable analogue atpgammas, and the resulting complexes w ...200011021930
streptavidin-based containment systems for genetically engineered microorganisms.the use of genetically modified microorganisms for environmental remediation continues to be debated. conditional lethal systems with tightly regulated gene expression can be used to contain released microorganisms and ameliorate some of the concerns about horizontal gene transfer. we have described streptavidin-based suicide systems to address these concerns and evaluated their function in pseudomonas putida containing the tol plasmid for aromatic hydrocarbon metabolism. tight regulation of exp ...199910796996
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
the xyls-dependent pm promoter is transcribed in vivo by rna polymerase with sigma 32 or sigma 38 depending on the growth phase.the pm promoter of the tol plasmid of pseudomonas putida is expressed at high level along the growth curve. this transcription is dependent on the positive regulator xyls activated by 3-methylbenzoate. the sigma factor sigma 38 is required for expression in early stationary phase and thereafter. to test whether sigma 70 was involved in pm transcription in exponential phase, we have followed mrna synthesis in a rpod thermosensitive strain. no difference in pm transcription was found between the w ...199910096078
adenosylcobalamin-mediated methyl transfer by toluate cis-dihydrodiol dehydrogenase of the tol plasmid pww0.we identified and characterized a methyl transfer activity of the toluate cis-dihydrodiol (4-methyl-3,5-cyclohexadiene-cis-1, 2-diol-1-carboxylic acid) dehydrogenase of the tol plasmid pww0 towards toluene cis-dihydrodiol (3-methyl-4,5-cyclohexadiene-cis-1, 2-diol). when the purified enzyme from the recombinant escherichia coli containing the xyll gene was incubated with toluene cis-dihydrodiol in the presence of nad+, the end products differed depending on the presence of adenosylcobalamin (coe ...199910217792
microscopic methods for distinguishing among three cell types in tol plasmid-carrying pseudomonas putida cultures.microscopic methods were developed that enable the sensitive quantification of different cell types that are generated by plasmid instability processes when pseudomonas putida paw164 (x+), which carries a tol plasmid (pww0-164), is grown in chemostat culture. cells that have lost the structural tol genes (x-) or the entire tol plasmid (x0) can be quantified in a background of 6000 x+ cells using catechol agarose miniplates. x0 cells can be quantified in a background of 3500 x+ or x- cells using ...199910220895
allylic or benzylic stabilization is essential for catalysis by bacterial benzyl alcohol dehydrogenases.benzyl alcohol dehydrogenase from acinetobacter calcoaceticus (ac-badh) and tol plasmid-encoded benzyl alcohol dehydrogenase from pseudomonas putida (tol-badh) have previously been shown to oxidize a variety of aromatic alcohols but not aliphatic substrates. here, we have expressed the genes for ac-badh and tol-badh in escherichia coli, purified the resulting over-expressed enzymes, and shown that each is an effective catalyst of both benzylic and allylic alcohol oxidation, but not of oxidation ...199910334943
the iiantr (ptsn) protein of pseudomonas putida mediates the c source inhibition of the sigma54-dependent pu promoter of the tol plasmid.the gene cluster adjacent to the sequence of rpon (encoding sigma factor sigma54) of pseudomonas putida has been studied with respect to the c source regulation of the pu promoter of the upper tol (toluene catabolism) operon. the region includes four open reading frames (orfs), two of which (named ptsn and ptso genes) encode proteins similar to components of the phosphoenolpyruvate:sugar phosphotransferase system. each of the four genes was disrupted with a nonpolar insertion, and the effects in ...199910336451
characterization of a pseudomonas putida allylic alcohol dehydrogenase induced by growth on 2-methyl-3-buten-2-ol.we have been working to develop an enzymatic assay for the alcohol 2-methyl-3-buten-2-ol (232-mb), which is produced and emitted by certain pines. to this end we have isolated the soil bacterium pseudomonas putida mb-1, which uses 232-mb as a sole carbon source. strain mb-1 contains inducible 3-methyl-2-buten-1-ol (321-mb) and 3-methyl-2-buten-1-al dehydrogenases, suggesting that 232-mb is metabolized by isomerization to 321-mb followed by oxidation. 321-mb dehydrogenase was purified to near-hom ...199910347052
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