Publications
| Title | Abstract | Year(sorted ascending) Filter | PMID Filter |
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| 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 |
| identification of the repressor subdomain within the signal reception module of the prokaryotic enhancer-binding protein xylr of pseudomonas putida. | in the presence of m-xylene, the protein xylr encoded by the tol plasmid of pseudomonas putida, activates the final sigma54-dependent promoter pu. early activation stages involve the release of the intramolecular repression caused by the signal reception n-terminal (a domain) of xylr on the central module of the protein. a genetic approach has been followed to locate the specific segment within a domain of xylr that is directly responsible for its down-regulation in the absence of inducer, as co ... | 1996 | 8626467 |
| catechol 2,3-dioxygenases functional in oxygen-limited (hypoxic) environments. | we studied the degradation of toluene for bacteria isolated from hypoxic (i.e., oxygen-limited) petroleum-contaminated aquifers and compared such strains with other toluene degraders. three pseudomonas isolates, p. pickettii pko1, pseudomonas sp. strain w31, and p. fluorescens cfs215, grew on toluene when nitrate was present as an alternate electron acceptor in hypoxic environments. we examined kinetic parameters (k(m) and vmax) for catechol 2,3-dioxygenase (c230), a key shared enzyme of the tol ... | 1996 | 8633871 |
| expression of the tol plasmid xyls gene in pseudomonas putida occurs from a alpha 70-dependent promoter or from alpha 70- and alpha 54-dependent tandem promoters according to the compound used for growth. | growth of pseudomonas putida (pwwo) on alkylbenzoates requires the expression of the meta pathway operon, which is mediated by the xyls protein after binding of a benzoate effector. alternatively, in cells growing on toluene or its aromatic alcohols, overexpression of xyls mediated by xylr activated by these compounds leads to overproduction of the xyls regulator, which even in the absence of benzoate effectors stimulates transcription from the meta cleavage pathway operon promoter. we show here ... | 1996 | 8636038 |
| physical and functional analysis of the prokaryotic enhancer of the sigma 54-promoters of the tol plasmid of pseudomonas putida. | the physical and the functional organization of the upstream cis-acting sequence that controls at a distance the transcriptional activity of pu and ps, the two sigma 54-dependent promoters of the tol (toluene/xylene biodegradation) operons of pseudomonas putida, have been determined. dnase i and hydroxyl radical footprinting of the promoters with the purified and pre-activated enhancer-binding protein xylr clearly indicated the presence of two distinct binding sites (proximal and distal) that we ... | 1996 | 8636992 |
| untranslated sequence upstream of mara in the multiple antibiotic resistance locus of escherichia coli is related to the effector-binding domain of the xyls transcriptional activator. | mara, the 129-amino-acid (aa) protein which plays a crucial role in the multiple antibiotic resistance (mar) phenotype in escherichia coli, shows homology to members of the xyls/arac family of transcriptional regulators. although these proteins vary in size from around 100 to 350 aa they all contain a dna-binding domain with a helix-turn-helix motif. the larger ones, e.g., xyls, arac, and rob, contain an additional domain either at their amino- or at their carboxyterminus. this domain is importa ... | 1996 | 8642609 |
| vtr expression cassettes for engineering conditional phenotypes in pseudomonas: activity of the pu promoter of the tol plasmid under limiting concentrations of the xylr activator protein. | a simplified procedure to construct recombinant pseudomonas putida (pp) and related bacteria, which transcribe conditionally specific genes inserted into their chromosome in response to lac inducers such as iptg, has been developed. the method is based on the so-called vtr expression cassettes. these are three small (1.98-kb) dna segments engineered as noti restriction fragments that include a laciq gene along with the hybrid trp/lac promoter, ptrc, followed by an optimised translation initiatio ... | 1996 | 8654996 |
| bacterial plasmid conjugation on semi-solid surfaces monitored with the green fluorescent protein (gfp) from aequorea victoria as a marker. | horizontal transfer of the tol plasmid was examined in pseudomonas putida (pp) kt2442 micro-colonies on semi-solid agar surfaces. horizontal gene transfer is usually studied in large populations where all information is based on average estimates of the transfer events in the entire population. we have used the green fluorescent protein (gfp) from the jellyfish aequorea victoria as a plasmid marker, in combination with single-cell observations. this provided hitherto unknown details on the distr ... | 1996 | 8707058 |
| 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 |
| sigma54-dependent transcription of the pseudomonas putida xyls operon is influenced by the iiantr protein of the phosphotransferase system in escherichia coli. | iiantr, encoded within the rpon operons of many gram-negative bacteria, is a homologue of a class of phosphoryl transfer proteins of the phosphoenolpyruvate: sugar phosphotransferase system. we have used a xyls operon-lacz fusion from the tol plasmid of pseudomonas putida to show that iiantr influences sigma 54-dependent transcription when the xyls operon is expressed in escherichia coli. loss of iiantr influences, but does not abolish cyclic amp-independent carbon catabolite repression. | 1996 | 8761731 |
| 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 |
| 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 |
| toluene elicits a carbon starvation response in pseudomonas putida mt-2 containing the tol plasmid pww0. | pseudomonas putida mt-2(pwwo) exhibited a carbon starvation response in the presence of toluene, a utilizable carbon source. when growth-supporting (4-mg/liter), inhibitory (130-mg/liter), and lethal (267-mg/ liter) levels of toluene were provided as the sole carbon source, p. putida responded by rapidly inhibiting protein synthesis and by producing 26 new proteins, 22 of which overlapped with those induced by carbon starvation. p. putida produced the same proteins when cultures were starved by ... | 1997 | 16535605 |
| 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 |
| construction and use of a versatile set of broad-host-range cloning and expression vectors based on the rk2 replicon. | the plasmid vectors described in this report are derived from the broad-host-range rk2 replicon and can be maintained in many gram-negative bacterial species. the complete nucleotide sequences of all of the cloning and expression vectors are known. important characteristics of the cloning vectors are as follows: a size range of 4.8 to 7.1 kb, unique cloning sites, different antibiotic resistance markers for selection of plasmid-containing cells, orit-mediated conjugative plasmid transfer, plasmi ... | 1997 | 9023917 |
| 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 |
| improved broad-host-range rk2 vectors useful for high and low regulated gene expression levels in gram-negative bacteria. | this report describes the construction and use of improved broad-host-range expression vectors based on the previously constructed pjb137 and pjb653 plasmids (blatny et al., 1997). these vectors contain the minimal replicon of rk2 and the inducible pu or pm promoters together with their regulatory xylr or xyls genes, respectively, from the pseudomonas putida tol plasmid pwwo. a set of atg vectors were derived from pjb653, and these vectors are characterized by the relatively small size, the pres ... | 1997 | 9281494 |
| 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 |
| cloning and sequence analysis of a catechol 2,3-dioxygenase gene from the nitrobenzene-degrading strain comamonas sp js765. | comamonas sp strain js765 utilizes nitrobenzene as a carbon and nitrogen source. the initial attack on nitrobenzene is carried out by nitrobenzene 1,2-dioxygenase, which converts nitrobenzene to an unstable nitrohydrodiol that spontaneously decomposes to form catechol and nitrite. catechol is then degraded via a meta cleavage pathway. we now report the cloning of a dna fragment carrying a catechol 2,3-dioxygenase gene from js765. nucleotide sequence analysis revealed three open reading frames (o ... | 1997 | 9451836 |
| modulation of the function of the signal receptor domain of xylr, a member of a family of prokaryotic enhancer-like positive regulators. | the xylr protein controls expression from the pseudomonas putida tol plasmid upper pathway operon promoter (pu) in response to aromatic effectors. xylr-dependent stimulation of transcription from a pu::lacz fusion shows different induction kinetics with different effectors. with toluene, activation followed a hyperbolic curve with an apparent k of 0.95 mm and a maximum beta-galactosidase activity of 2,550 miller units. with o-nitrotoluene, in contrast, activation followed a sigmoidal curve with ... | 1998 | 9457863 |
| in situ gene expression in mixed-culture biofilms: evidence of metabolic interactions between community members. | microbial communities growing in laboratory-based flow chambers were investigated in order to study compartmentalization of specific gene expression. among the community members studied, the focus was in particular on pseudomonas putida and a strain of an acinetobacter sp., and the genes studied are involved in the biodegradation of toluene and related aromatic compounds. the upper-pathway promoter (pu) and the meta-pathway promoter (pm) from the tol plasmid were fused independently to the gene ... | 1998 | 9464414 |
| 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 |
| development and testing of a bacterial biosensor for toluene-based environmental contaminants. | a bacterial biosensor for benzene, toluene, and similar compounds has been constructed, characterized, and field tested on contaminated water and soil. the biosensor is based on a plasmid incorporating the transcriptional activator xylr from the tol plasmid of pseudomonas putida mt-2. the xylr protein binds a subset of toluene-like compounds and activates transcription at its promoter, pu. a reporter plasmid was constructed by placing the luc gene for firefly luciferase under the control of xylr ... | 1998 | 9501440 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| cloning and nucleotide sequence of carbazole catabolic genes from pseudomonas stutzeri strain om1, isolated from activated sludge. | a new carbazole (car)-degrading bacterium, called strain om1, was isolated from activated sludge obtained from sewage disposal plants in fukuoka prefecture, and it was identified as pseudomonas stutzeri. anthranilic acid (an), 2'-aminobiphenyl-2,3-diol and its meta-cleavage product, 2-hydroxy-6-oxo-6-(2'-aminophenyl)-hexa-2,4-dienoic acid, were identified as metabolic intermediates of car in the ethyl acetate extract of the culture broth. therefore, the car catabolic pathway to an in strain om1 ... | 1998 | 12501294 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 ... | 2002 | 12534468 |
| 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 ... | 2003 | 12547188 |
| 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 ... | 2003 | 12754236 |
| 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 |
| 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 ... | 2003 | 12902279 |
| 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 ... | 2003 | 12906358 |
| 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 ... | 2003 | 14630048 |
| 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 ... | 2004 | 14672954 |
| 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 ... | 2004 | 14759656 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 ... | 2006 | 16584471 |
| 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 ... | 2006 | 16689797 |
| 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 |
| 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 |