| the specific growth rate of pseudomonas putida paw1 influences the conjugal transfer rate of the tol plasmid. | the kinetics of the conjugal transfer of a tol plasmid were investigated by using pseudomonas putida paw1 as the donor strain and p. aeruginosa pao 1162 as the recipient strain. short-term batch mating experiments were performed in a nonselective medium, while the evolution of the different cell types was determined by selective plating techniques. the experimental data were analyzed by using a mass action model that describes plasmid transfer kinetics. this method allowed analysis of the mating ... | 1993 | 8250565 |
| new derivatives of tol plasmid pww0. | two new segregants, ppw1-1 and ppw161-1, of pseudomonas putida were isolated from the stock cultures paw85(pww0) and paw85(pww0-161). strain ppw1-1 had lost its ability to grow on m-xylene but was able to grow on m-toluate. a deletion of the left-hand of transposon tn4651, including the upper-operon genes, had taken place in plasmid pww0mut1, isolated from strain ppw1-1. additional deletions were observed in pww0mut1 after 'benzoate-curing' (plasmids pww0mut15, pww0mut19, pww0mut27). the genes o ... | 1993 | 8254307 |
| effect of bacterial distribution and activity on conjugal gene transfer on the phylloplane of the bush bean (phaseolus vulgaris). | conjugal plasmid transfer was examined on the phylloplane of bean (phaseolus vulgaris) and related to the spatial distribution pattern and metabolic activity of the bacteria. the donor (pseudomonas putida kt2442) harbored a derivative of the tol plasmid, which conferred kanamycin resistance and had the gfp gene inserted downstream of a lac promoter. a chromosomal insertion of laciq prevented expression of the gfp gene. the recipient (p. putida kt2440) had a chromosomal tetracycline resistance ma ... | 1998 | 9572970 |
| in vivo reactivation of catechol 2,3-dioxygenase mediated by a chloroplast-type ferredoxin: a bacterial strategy to expand the substrate specificity of aromatic degradative pathways. | the meta-cleavage operon of the tol plasmid pww0 of pseudomonas putida contains 13 genes responsible for the oxidation of benzoate and toluates to krebs cycle intermediates via estradiol (meta) cleavage of (methyl)catechol. the functions of all the genes are known with the exception of xylt. we constructed pww0 mutants defective in the xylt gene, and found that these mutants were not able to grow on p-toluate while they were still capable of growing on benzoate and m-toluate. in the xylt mutants ... | 1993 | 8344270 |
| tetrameric structure and cellular location of catechol 2,3-dioxygenase. | catechol 2,3-dioxygenase from the meta-cleavage pathway encoded on the tol plasmid of pseudomonas putida (pwwo) was investigated by electron microscopy. negatively stained samples of the purified catechol 2,3-dioxygenase revealed that the enzyme consists of four subunits arranged in a tetrahedral conformation. monoclonal antibodies raised against catechol 2,3-dioxygenase showed highly specific reactions and were used to localize the enzyme in escherichia coli (paw31) and p. putida (pwwo), using ... | 1995 | 7710322 |
| cold shock proteins and cold acclimation proteins in the psychrotrophic bacterium pseudomonas putida q5 and its transconjugant. | the production of cold shock proteins (csps) and cold acclimation proteins (caps) was characterized in the psychrotrophic bacterium pseudomonas putida q5 and its transconjugant p. putida q5t which contains the toluene-degradative tol (pwwo) plasmid, using two-dimensional gel electrophoresis and computing scanning laser densitometry. similar growth rates for the psychrotrophic bacterium p. putida q5 and the transconjugant were found at temperatures ranging from 30 to 0 degree c. sixteen proteins ... | 1996 | 8776850 |
| involvement of sigma 54 in exponential silencing of the pseudomonas putida tol plasmid pu promoter. | the sigma 54-dependent pu promoter of the tol plasmid pww0 of pseudomonas putida becomes activated by the prokaryotic enhancer-binding xyir protein when cells encounter m-xylene in the medium. however, even in the presence of the aromatic inducer, pu activity is silenced in vivo during rapid exponential growth of the cells in rich medium. various elements known to be involved in the control of the transcriptional activity of the promoter were examined to ascertain the mechanism by which expressi ... | 1996 | 8821932 |
| role of sigma s in transcription from the positively controlled pm promoter of the tol plasmid of pseudomonas putida. | transcription from the tol plasmid pm promoter is dependent on the xyls regulator activated by benzoate effectors. we analysed transcription from pm in several backgrounds with differing escherichia coli alpha and sigma subunits of rna polymerase. in different rpoa backgrounds, transcription from pm was as high as in the wild-type background throughout the growth curve. in the sigma s-deficient background provided by e. coli rh90, high levels of transcription from pm (xyls/3-methylbenzoate depen ... | 1995 | 8825089 |
| structure and function of the pseudomonas putida integration host factor. | integration host factor (ihf) is a dna-binding and -bending protein that has been found in a number of gram-negative bacteria. here we describe the cloning, sequencing, and functional analysis of the genes coding for the two subunits of ihf from pseudomonas putida. both the ihfa and ihfb genes of p. putida code for 100-amino-acid-residue polypeptides that are 1 and 6 residues longer than the escherichia coli ihf subunits, respectively. the p. putida ihfa and ihfb genes can effectively complement ... | 1996 | 8892836 |
| the tacan4tgca motif upstream from the -35 region in the sigma70-sigmas-dependent pm promoter of the tol plasmid is the minimum dna segment required for transcription stimulation by xyls regulators. | transcription from the tol plasmid meta-cleavage pathway operon promoter pm is dependent on the xyls regulator activated by benzoate effectors or after xyls overproduction. we have generated 5' deletions in pm and have analyzed expression from wild-type and mutant promoters with the wild-type xyls regulator and xyls mutant regulators that stimulated transcription constitutively. we have found that the motifs t(c or a)can4tgca located between -46 and -57 and -67 and -78 with respect to the main t ... | 1996 | 8932297 |
| organisation of the tmb catabolic operons of pseudomonas putida tmb and evolutionary relationship with the xyl operons of the tol plasmid pww0. | in pseudomonas putida (pp) tmb the genes involved in the catabolism of methyl-substituted aromatic hydrocarbons 1,2,4-trimethylbenzene, m- and p-xylene (tmb operon), are functionally and genetically homologous to the xyl genes of the plasmid pww0, but are chromosomally encoded. we have analysed by cloning. southern blotting and sequencing of selected regions the organisation of the tmb cluster. this analysis shows that the structural and regulatory genes of the tmb and xyl systems exhibit a high ... | 1996 | 8982087 |
| relative expression and stability of a chromosomally integrated and plasmid-borne marker gene fusion in environmentally competent bacteria. | a xyle-icec transcriptional fusion was created by ligatinga dna fragment harboring the cloned xyle structural gene from the tol plasmid of pseudomonas putida mt-2 into the cloned icec gene of pseudomonas syringae cit7. this fusion construct was integrated into the chromosome of pseudomonas syringae cit7 by homologous recombination. both cis-merodiploid strain cit7m17 and marker exchange strain cit7h69 produced the xyle gene product, catechol2,3-dioxygenase. strain cit7m17, in which xyle was infl ... | 1997 | 9003582 |
| characterization of the p-toluenesulfonate operon tsambcd and tsar in comamonas testosteroni t-2. | comamonas testosteroni t-2 uses a standard, if seldom examined, attack on an aromatic compound and oxygenates the side chain of p-toluenesulfonate (ts) (or p-toluenecarboxylate) to p-sulfobenzoate (or terephthalate) prior to complete oxidation. the expression of the first three catabolic enzymes in the pathway, the ts methyl-monooxygenase system (comprising reductase b and oxygenase m; tsamb), p-sulfobenzyl alcohol dehydrogenase (tsac), and p-sulfobenzaldehyde dehydrogenase (tsad), is coregulate ... | 1997 | 9006050 |
| xyluw, two genes at the start of the upper pathway operon of tol plasmid pww0, appear to play no essential part in determining its catabolic phenotype. | the upper pathway operon of the toluene catabolic pathway of tol plasmid pww0 was shown to carry two open reading frames between the start of transcription and xylc (encoding benzaldehyde dehydrogenase), the first previously reported gene of the operon. these were designated xyluw: xylu encoded a protein of 131 amino acid residues (m(r) 14,244) which bore no relationship with any protein in the databases, and xylw encoded a protein of 348 residues (m(r) 36,992) which was strongly homologous to o ... | 1997 | 9025283 |
| 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 ... | 1997 | 9098077 |
| genetic evidence of separate repressor and activator activities of the xylr regulator of the tol plasmid, pww0, of pseudomonas putida. | the xylr protein encoded by pww0, the tol (toluene biodegradation) plasmid of pseudomonas putida, activates at a distance the transcription of pu and ps, which are the two sigma(54)-dependent promoters of the plasmid, but it also downregulates its own sigma(70)-promoter, pr, which divergently overlaps the upstream activating sites of ps. all regulatory elements that control pr activity have been faithfully reproduced in escherichia coli, and the basis of the autoregulation of xylr transcription ... | 1997 | 9106213 |
| fiber-optic-based biomonitoring of benzene derivatives by recombinant e. coli bearing luciferase gene-fused tol-plasmid immobilized on the fiber-optic end. | tol plasmid in pseudomonas putida mt-2 has a series of genes for the degradation of xylene, toluene, and their derivatives to pyruvate and acetaldehyde (or propionaldehyde). two operons, i.e., upper operon and meta operon, play indispensable roles for the digestion of xylene derivatives: when xyir protein recognizes xylene derivatives, another controlling gene, xyis, is activated, which results in the activation of meta operon. therefore, we have constructed a fusion gene between tol plasmid and ... | 1997 | 9212714 |
| 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 ... | 1997 | 9260942 |
| transcriptional control of the pseudomonas tol plasmid catabolic operons is achieved through an interplay of host factors and plasmid-encoded regulators. | the xyl genes of pseudomonas putida tol plasmid that specify catabolism of toluene and xylenes are organized in four transcriptional units: the upper-operon xyluwcambn for conversion of toluene/xylenes into benzoate/alkylbenzoates; the meta-operon xylxyzltegfjqkih, which encodes the enzymes for further conversion of these compounds into krebs cycle intermediates; and xyls and xylr, which are involved in transcriptional control. the xyls and xylr proteins are members of the xyls/arac and ntrc fam ... | 1997 | 9343354 |
| activation of the toluene-responsive regulator xylr causes a transcriptional switch between sigma54 and sigma70 promoters at the divergent pr/ps region of the tol plasmid. | the mechanism by which xylr, the toluene-responsive activator of the sigma54-dependent pu and ps promoters of the pseudomonas tol plasmid pww0, downregulates its own sigma70 promoter prhas been examined. an in vitro transcription system was developed in order to reproduce the repression of probserved in cells of p. putida (pww0) both in the presence and in the absence of the xylr inducer, benzyl alcohol. dna templates bearing the two sigma70-rna polymerase (rnap) binding sites of pr, which overl ... | 1998 | 9489676 |
| molecular characterization of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase ii of acinetobacter calcoaceticus. | the nucleotide sequences of xylb and xylc from acinetobacter calcoaceticus, the genes encoding benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase ii, were determined. the complete nucleotide sequence indicates that these two genes form part of an operon and this was supported by heterologous expression and physiological studies. benzaldehyde dehydrogenase ii is a 51654 da protein with 484 amino acids per subunit and it is typical of other prokaryotic and eukaryotic aldehyde dehydrogenas ... | 1998 | 9494109 |
| 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 ... | 1999 | 10796996 |
| 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 ... | 2000 | 10809692 |
| 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 ... | 1998 | 9521650 |
| 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 ... | 2000 | 10913634 |
| a novel -2fe-2s- ferredoxin from pseudomonas putida mt2 promotes the reductive reactivation of catechol 2,3-dioxygenase. | catechol 2,3-dioxygenase (xyle) is a component of the tol plasmid-encoded pathway for the degradation of toluene and xylenes and catalyzes the dioxygenolytic cleavage of the aromatic ring. purified xyle is oxygen-sensitive and unstable in vitro, particularly in the presence of substituted catechol substrates, but it is stabilized in vivo by another protein, xylt, encoded by the xylt gene located just upstream of xyle. in this study, we have purified to homogeneity the xylt product from a recombi ... | 1998 | 9545294 |
| 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 ... | 1996 | 8713131 |
| tol plasmid transcription factor xyls binds specifically to the pm operator sequence. | xyls, an arac family transcription factor, positively regulates transcription of pseudomonas putida tol plasmid meta operon from the pm promoter. a tandem of 15 bp homologous direct repeats, separated by 6 bp and overlapping with the -35 hexamer of the promoter, is required for the activation of pm by xyls in vivo. in this study we have characterized specific binding of xyls to the pm operator om. xyls was overexpressed with an epitope tag in its n-terminus. tagged xyls (n-xyls) was immunopurifi ... | 1996 | 8736536 |
| genetic evidence for activation of the positive transcriptional regulator xy1r, a member of the ntrc family of regulators, by effector binding. | the xy1r protein positively controls expression from the pseudomonas putida tol plasmid sigma 54-dependent pu and ps promoters, in response to the presence of aromatic effectors such as m-xylene, m-methylbenzyl alcohol, and p-chlorobenzaldehyde in the culture medium. xy1r also autoregulates its own synthesis. a mutant xy1r regulator called xy1r7 was isolated after nitrosoguanidine mutagenesis of the wild-type gene and phenotypic selection for mutants that had acquired the ability to recognize m- ... | 1994 | 8132529 |
| implications of the xylq gene of tol plasmid pww102 for the evolution of aromatic catabolic pathways. | pseudomonas putida strain o2c2 is able to grow on toluene, m-xylene and p-xylene through benzoate and the corresponding methylbenzoates (toluates). the catabolic genes are encoded on a large tol plasmid, pww102, of > 220 kb. the complete catabolic genes were cloned on four large overlapping restriction fragments covering a total of 28 kb of the plasmid, which was carefully mapped by restriction enzyme analysis. the presence of the xyl genes on the cloned dna was confirmed by assay of representat ... | 1998 | 9611813 |
| purification, characterization, and gene analysis of catechol 2,3-dioxygenase from the aniline-assimilating bacterium pseudomonas species aw-2. | catechol 2,3-dioxygenase (c23d; ec 1.13.1.2) was purified to homogeneity from a cell extract of pseudomonas sp. aw-2 grown on aniline, and the purified c23d was characterized. the molecular mass estimated by gel filtration was 110 kda. the enzyme dissociated into four identical subunits each with the molecular mass of 33 kda. the enzyme had high activity for 3-methylcatechol as well as catechol, and differed from the enzyme from pseudomonas putida mt-2, which carries the tol plasmid, in optimal ... | 1998 | 9614705 |
| the rpos gene regulates op2, an operon for the lower pathway of xylene catabolism on the tol plasmid, and the stress response in pseudomonas putida mt-2. | operon op2 on the pseudomonas putida tol plasmid encodes enzymes for m-toluate catabolism; transcription of this operon is activated by xyls in the presence of m-toluate. because transcriptional activation of op2 specifically occurs in the stationary phase of growth both in p. putida and in escherichia coli, we suspected that its transcription is dependent on rpos (sigmas). therefore, we constructed a rpos disruption strain of p. putida mt-2, and assayed op2 expression and other phenotypes. op2 ... | 1998 | 9738882 |
| protein binding in vivo to op2 promoter of the pseudomonas putida tol plasmid. | the transcription of op2 encoding enzymes for m-toluate catabolism on the pseudomonas putida tol plasmid is activated by basal-level xyls protein in the presence of m-toluate or by overproduced xyls protein in the absence of m-toluate. in this study, in vivo dimethyl sulfate (dms) footprinting was performed to understand the mechanism of transcriptional regulation of op2 promoter by xyls. in the presence of overproduced xyls without m-toluate, several protected nucleotides were observed, indicat ... | 1998 | 9861447 |
| 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 ... | 1999 | 9890992 |
| 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 ... | 1999 | 10096078 |
| 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 ... | 2001 | 11591664 |
| 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 ... | 1999 | 10217792 |
| 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 ... | 1999 | 10220895 |
| 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 ... | 1999 | 10334943 |
| 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 ... | 1999 | 10336451 |
| 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 ... | 1999 | 10347052 |
| 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 ... | 2002 | 12426347 |
| catalytic properties of the 3-chlorocatechol-oxidizing 2, 3-dihydroxybiphenyl 1,2-dioxygenase from sphingomonas sp. strain bn6. | the 2,3-dihydroxybiphenyl dioxygenase from sphingomonas sp. strain bn6 (bphc1-bn6) differs from most other extradiol dioxygenases by its ability to oxidize 3-chlorocatechol to 3-chloro-2-hydroxymuconic semialdehyde by a distal cleavage mechanism. the turnover of different substrates and the effects of various inhibitors on bphc1-bn6 were compared with those of another 2,3-dihydroxybiphenyl dioxygenase from the same strain (bphc2-bn6) as well as with those of the archetypical catechol 2,3-dioxyge ... | 1999 | 10438749 |
| 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 ... | 2000 | 10640666 |
| 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 ... | 2000 | 10648520 |
| 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 ... | 2000 | 10648539 |
| 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 ... | 2003 | 12788725 |
| ntn genes determining the early steps in the divergent catabolism of 4-nitrotoluene and toluene in pseudomonas sp. strain tw3. | pseudomonas sp. strain tw3 is able to oxidatively metabolize 4-nitrotoluene and toluene via a route analogous to the upper pathway of the tol plasmids. we report the sequence and organization of five genes, ntnwcmab*, which are very similar to and in the same order as the xyl operon of tol plasmid pww0 and present evidence that they encode enzymes which are expressed during growth on both 4-nitrotoluene and toluene and are responsible for their oxidation to 4-nitrobenzoate and benzoate, respecti ... | 1998 | 9555884 |
| regulation of benzoate degradation in acinetobacter sp. strain adp1 by benm, a lysr-type transcriptional activator. | in acinetobacter sp. strain adp1, benzoate degradation requires the ben genes for converting benzoate to catechol and the cat genes for degrading catechol. here we describe a novel transcriptional activator, benm, that regulates the chromosomal ben and cat genes. benm is homologous to catm, a lysr-type transcriptional activator of the cat genes. unusual regulatory features of this system include the abilities of both benm and catm to recognize the same inducer, cis,cis-muconate, and to regulate ... | 1998 | 9573203 |
| 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 ... | 1998 | 9603843 |
| activation and repression of transcription at the double tandem divergent promoters for the xylr and xyls genes of the tol plasmid of pseudomonas putida. | the xylr and xyls genes are divergent and control transcription of the tol plasmid catabolic pathways for toluene metabolism. four promoters are found in the 300-bp intergenic region: pr1 and pr2 are constitutive sigma70-dependent tandem promoters that drive expression of xylr, while expression of the xyls gene is driven from ps2, a constitutive sigma70-dependent promoter, and by the regulatable sigma54 class ps1 promoter. in ps1 the xylr targets (upstream activator sequences [uass]) overlap the ... | 1998 | 9603877 |
| 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 ... | 2001 | 11179677 |
| 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 ... | 2000 | 11200433 |
| 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 ... | 2001 | 11375176 |
| 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 ... | 2005 | 15640169 |
| 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 ... | 2001 | 11462134 |
| 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 ... | 2006 | 16055251 |
| 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 ... | 2005 | 16085802 |
| 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 ... | 2005 | 16135224 |
| 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 ... | 2001 | 11472911 |
| 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 ... | 2001 | 11673437 |
| novel alterations in plasmid dna associated with aromatic hydrocarbon utilization by pseudomonas putida r5-3. | subcultures of pseudomonas putida r5-3 altered their plasmid dna content in specific ways depending on the particular aromatic hydrocarbon utilized as the sole carbon source. two indigenous plasmids, 115 and 95 kilobases (kb) in size, were observed in r5-3a, which was derived from r5-3 by growth on minimal medium containing p-methylbenzoate as the sole carbon source. when r5-3a was transferred to medium containing m-xylene or toluene, derivative strains were obtained in which the 95-kb plasmid w ... | 1989 | 16347946 |
| 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. ... | 2001 | 11679066 |
| 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 ... | 2002 | 11846413 |
| 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 ... | 2006 | 16420611 |
| 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 ... | 2006 | 16510445 |
| 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 ... | 2002 | 12023029 |
| 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 ... | 2002 | 12297005 |
| 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 ... | 2000 | 10850662 |
| 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 ... | 2000 | 10940009 |
| 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 ... | 2000 | 11021930 |
| 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 ... | 2001 | 11160806 |
| 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 ... | 2001 | 11160899 |
| 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 ... | 2001 | 11165359 |
| 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 ... | 2004 | 15576775 |
| 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 ... | 2007 | 17504492 |
| 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 ... | 2005 | 15601696 |
| 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 ... | 2008 | 18005985 |
| 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 ... | 2008 | 18076969 |
| 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 ... | 2005 | 16030214 |
| 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 ... | 2005 | 16332855 |
| plasmid incidence in bacteria from deep subsurface sediments. | bacteria were isolated from deep terrestrial subsurface sediments underlying the coastal plain of south carolina. a total of 163 isolates from deep sediments, surface soil, and return drill muds were examined for plasmid dna content and resistance to the antibiotics penicillin, ampicillin, carbenicillin, streptomycin, kanamycin, and tetracycline. mics of cu, cr, and hg for each isolate were also determined. the overall frequency of plasmid occurrence in the subsurface bacteria was 33%. resistanc ... | 1988 | 16347789 |
| biosynthesis of synthons in two-liquid-phase media. | the pseudomonas oleovorans alkane hydroxylase and xylene oxygenase from pseudomonas putida are versatile mono-oxygenases for stereo- and regioselective oxidation of aliphatic and aromatic hydrocarbons. pseudomonas oleovorans and alkanol dehydrogenase deficient mutants of pseudomonas have previously been used to produce alkanols from various alkanes and optically active epoxides from alkenes. similarly, p. putida strains have been used to produce aromatic alcohols, aromatic acids, and optically a ... | 1996 | 18629897 |
| conditional-suicide containment system for bacteria which mineralize aromatics. | a model conditional-suicide system to control genetically engineered microorganisms able to degrade substituted benzoates is reported. the system is based on two elements. one element consists of a fusion between the promoter of the pseudomonas putida tol plasmid-encoded meta-cleavage pathway operon (p(m)) and the laci gene encoding lac repressor plus xyls, coding for the positive regulator of p(m). the other element carries a fusion between the p(tac) promoter and the gef gene, which encodes a ... | 1991 | 16348490 |
| 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 ... | 2008 | 19138193 |
| 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 ... | 2009 | 19176233 |
| 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, ... | 2009 | 19557171 |
| 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 ... | 2004 | 19712397 |
| nitroaromatics are substrates for the tol plasmid upper-pathway enzymes. | expression of the xylma genes encoding for toluene monoxygenase from the lactose promoter in a broad-host-range plasmid allows the oxidation of toluene and m- and p-nitrotoluene to their corresponding benzyl alcohols and benzaldehydes in pseudomonas putida and escherichia coli. benzyl alcohols accumulate until reaching a concentration of about 80 mum, while benzaldehydes accumulate steadily with time for at least 24 h. tol-encoded benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase recog ... | 1992 | 16348637 |
| 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 ... | 2010 | 20506384 |
| impact of growth in benzoate and m-toluate liquid media on culturability of pseudomonas putida on benzoate and m-toluate plates. | pseudomonas putida grown in continuous culture on benzoate or m-toluate lost the ability to grow on benzoate or m-toluate plates. a similar effect was not seen with a glucose continuous culture. cells carrying and expressing a tol plasmid rapidly lost their ability to grow on benzoate solid medium. | 1995 | 16535111 |
| 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 ... | 2010 | 20581207 |
| 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 ... | 2010 | 20709833 |
| 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 ... | 2010 | 20846143 |
| 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 ... | 2010 | 20872219 |
| 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 ... | 2010 | 21091520 |
| 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 ... | 2011 | 21205010 |
| 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 ... | 2011 | 22078029 |
| 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 ... | 2011 | 21947012 |
| 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 ... | 2013 | 23658554 |