Publications

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
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
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
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
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
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
amplification of toluene dioxygenase genes in a hybrid pseudomonas strain to enhance the biodegradation of benzene, toluene, and p-xylene mixture.	a hybrid metabolic pathway through which benzene, toluene, and p-xylene (btx) mixture could be simultaneously mineralized was previously constructed in pseudomonas putida tb101 (lee, roh, kim, biotechnol. bioeng 43: 1146-1152, 1994). in this work, we improved the performance of the hybrid pathway by cloning the todc1c2ba genes in the broad-host-range multicopy vector rsf1010 and by introducing the resulting plasmid ptol037 into p. putida mt-2 which harbors the archetypal tol plasmid. as a result ...	1995	18623248
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
construction and behavior of biologically contained bacteria for environmental applications in bioremediation.	the survival of microorganisms can be predicted through the use of active biological containment systems. we have constructed contained pseudomonas putida strains that degrade alkylbenzoates. the modified strain carries a fusion of the plac promoter to the gef gene, which encodes a killing protein. expression from plac is controlled through a regulatory cascade, so that plac is switched on or off by the absence or presence of alkylbenzoates, respectively. similar uncontained strains were also co ...	1995	7487030
the 4-hydroxy-2-oxovalerate aldolase and acetaldehyde dehydrogenase (acylating) encoded by the nahm and naho genes of the naphthalene catabolic plasmid pww60-22 provide further evidence of conservation of meta-cleavage pathway gene sequences.	we report the complete nucleotide sequence and over-expression of the nahom genes for the acetaldehyde dehydrogenase (acylating) and the 4-hydroxy-2-oxovalerate aldolase from the meta pathway operon of the naphthalene catabolic plasmid pww60-22 from pseudomonas sp. ncimb9816. additional partial sequence analysis of adjacent dna shows the gene order within the operon to be nahnlomk, identical to the order found for the isofunctional genes in the meta pathway operons in the toluene/xylene pathway ...	1995	7496535
quantification of the effect of substrate concentration on the conjugal transfer rate of the tol plasmid in short-term batch mating experiments.	batch mating experiments with pseudomonas putida paw 1 (tol) as a donor and pseudomonas aeruginosa pao 1162 as a recipient strain were performed to quantify the effect of the substrate concentration in the mating medium on the observed plasmid transfer rate coefficient. the impact of the substrate concentration in the mating medium was highly correlated with the growth history of the donor strain. when the donor strain was harvested in exponential growth phase, no impact was observed; when the d ...	1995	7576502
the sigma 54-dependent promoter ps of the tol plasmid of pseudomonas putida requires hu for transcriptional activation in vivo by xylr.	in the presence of toluene and xylenes, the sigma 54-dependent ps promoter of the tol (toluene biodegradation) plasmid pww0 of pseudomonas putida is activated at a distance by the xylr protein, of the ntrc family of transcriptional regulators. since contacts between xylr bound to upstream activating sites and the rna polymerase require the looping out of the intervening dna segment, the intrinsic curvature, the bendability of the corresponding sequence, and the spatial effects of protein-induced ...	1995	7601841
isolation and expansion of the catabolic potential of a pseudomonas putida strain able to grow in the presence of high concentrations of aromatic hydrocarbons.	pseudomonas putida dot-t1 was isolated after enrichment on minimal medium with 1% (vol/vol) toluene as the sole c source. the strain was able to grow in the presence of 90% (vol/vol) toluene and was tolerant to organic solvents whose log p(ow) (octanol/water partition coefficient) was higher than 2.3. solvent tolerance was inducible, as bacteria grown in the absence of toluene required an adaptation period before growth restarted. mg2+ ions in the culture medium improved solvent tolerance. elect ...	1995	7608060
biosensing of benzene derivatives in the environment by luminescent escherichia coli.	sensitive and convenient biosensing of environmental pollutants has been developed by fusing a gene of firefly luciferase to the tol plasmid. tol plasmid of pseudomonas putida encodes a series of enzymes for degradation of benzene and its derivatives. the expression of these enzymes is controlled with the regulating proteins xylr and xyls, whose promoters are activated in the presence of aromatic compounds. the structural gene of firefly luciferase, as a reporter enzyme, was inserted under the c ...	1995	7612210
overexpression of pseudomonas putida catechol 2,3-dioxygenase with high specific activity by genetically engineered escherichia coli.	the cloned xyle gene encoding catechol 2,3-dioxygenase (metapyrocatechase) from tol plasmid in pseudomonas putida mt-2 has been expressed in escherichia coli w3110 to a level of approximately 15% of the total soluble protein. of the total iron in the crude extract, 45% was on the enzyme. the crystallized enzyme from e. coli had higher iron content (3.7 mol/mol enzyme) and specific activity (536 u/mg) than the enzyme from p. putida mt-2. however, no differences were observed in physicochemical, p ...	1995	7629031
integration host factor suppresses promiscuous activation of the sigma 54-dependent promoter pu of pseudomonas putida.	in the presence of m-xylene, the pu promoter of the tol plasmid of pseudomonas putida is activated by the prokaryotic enhancer-binding protein xylr. the intervening dna segment between the upstream activating sequences (uass) and those for rna polymerase binding contains an integration host factor (ihf) attachment site that is required for full transcriptional activity. in the absence of ihf, the pu promoter can be cross-activated by other members of the sigma 54-dependent family of regulatory p ...	1995	7638181
cloning and sequencing of the catechol 2,3-dioxygenase gene of alcaligenes sp. kf711.	the catechol 2,3-dioxygenase is an aromatic ring-fission enzyme catalyzing the conversion of catechol to 2-hydroxymuconic semialdehyde. a catechol 2,3-dioxygenase gene has been cloned from chromosomal dna of alcaligenes sp. kf711, and its sequence was determined. the catechol 2,3-dioxygenase gene was consisted of 927 nucleotides with atg initiation codon and tga termination codon, which can encode a polypeptide of molecular weight 35 kda containing 308 amino acid residues. g+c content of the gen ...	1995	7702624
tetrameric structure and cellular location of catechol 2,3-dioxygenase.	catechol 2,3-dioxygenase from the meta-cleavage pathway encoded on the tol plasmid of pseudomonas putida (pwwo) was investigated by electron microscopy. negatively stained samples of the purified catechol 2,3-dioxygenase revealed that the enzyme consists of four subunits arranged in a tetrahedral conformation. monoclonal antibodies raised against catechol 2,3-dioxygenase showed highly specific reactions and were used to localize the enzyme in escherichia coli (paw31) and p. putida (pwwo), using ...	1995	7710322
substrate specificity differences between two catechol 2,3-dioxygenases encoded by the tol and nah plasmids from pseudomonas putida.	the substrate specificities of two catechol 2,3-dioxygenases, one encoded by xyle on the tol plasmid pww0 and the other encoded by nahh on the nah7 plasmid, were investigated. the xyle catechol 2,3-dioxygenase catalyzes the ring-cleavage of catechol, 3-methylcatechol and 4-methylcatechol. the nahh catechol 2,3-dioxygenase was partially deficient in oxidizing 3-methylcatechol due to defects in two catalytic properties. first, nahh has a lower kcat value for 3-methylcatechol compared to xyle, and ...	1995	7744021
molecular and biochemical characterization of two meta-cleavage dioxygenases involved in biphenyl and m-xylene degradation by beijerinckia sp. strain b1.	beijerinckia sp. strain b1 is able to grow on either biphenyl or m-xylene as the sole source of carbon and is capable of cooxidizing many polycyclic aromatic hydrocarbons. the catabolic pathways for biphenyl and m-xylene degradation are coinduced and share common downstream enzymatic reactions. the catabolic pathway for biphenyl degradation involves two meta-cleavage steps, one for 2,3-dihydroxybiphenyl and a second for catechol. the catabolic pathway for m-xylene involves one m-cleavage step fo ...	1995	7768806
involvement of ihf protein in expression of the ps promoter of the pseudomonas putida tol plasmid.	regulation of the xyl gene operons of the pseudomonas putida tol plasmid is mediated by the products of the downstream clustered and divergently oriented xylr and xyls regulatory genes. the xylr-xyls intergenic region contains the xylr and xyls promoters pr and ps, respectively. a binding site for the xylr activator protein is located upstream of ps and overlapping pr. dnase i footprint experiments showed that one of these sites, which overlaps the recognition site for xylr activator, as well as ...	1995	7768832
overlapping substrate specificities of benzaldehyde dehydrogenase (the xylc gene product) and 2-hydroxymuconic semialdehyde dehydrogenase (the xylg gene product) encoded by tol plasmid pww0 of pseudomonas putida.	two aldehyde dehydrogenases involved in the degradation of toluene and xylenes, namely, benzaldehyde dehydrogenase and 2-hydroxymuconic semialdehyde dehydrogenase, are encoded by the xylc and xylg genes, respectively, on tol plasmid pww0 of pseudomonas putida. the nucleotide sequence of xylc was determined in this study. a protein exhibiting benzaldehyde dehydrogenase activity had been purified from cells of p. putida (pww0) (j. p. shaw and s. harayama, eur. j. biochem. 191:705-714, 1990); howev ...	1995	7868591
single amino acids changes in the signal receptor domain of xylr resulted in mutants that stimulate transcription in the absence of effectors.	the xylr protein positively controls expression from the pseudomonas putida tol plasmid sigma 54-dependent "upper" pathway operon promoter (pu) and the xyls gene promoter (ps), in response to the presence of aromatic effectors. two mutant xylr regulators able to stimulate transcription from pu and ps in the absence of effectors were isolated. these mutants exhibited single point mutations, namely asp135-->asn and pro85-->ser. both mutations are located in the amino termini domain of xylr, which ...	1995	7890623
identification of functional residues in a 2-hydroxymuconic semialdehyde hydrolase. a new member of the alpha/beta hydrolase-fold family of enzymes which cleaves carbon-carbon bonds.	the 2-hydroxymuconic semialdehyde hydrolase, xylf, of the pseudomonas putida tol plasmid-encoded pathway for the catabolism of toluene and xylenes, catalyzes one of the rarest types of enzyme reaction (ec 3.7.1.9), the hydrolysis of a carbon-carbon bond in its substrate, the ring-fission product of 3-alkyl-substituted catechols. in this study, amino acid sequence comparisons between xylf and other hydrolases, and analysis of the similarity between the predicted secondary structure of xylf and th ...	1995	7890778
analysis of dna bend structure of promoter regulatory regions of xylene-metabolizing genes on the pseudomonas tol plasmid.	the transcription of both the upper operon (op1) coding for m-xylene-degrading enzymes and the positive regulatory gene xyls on the tol plasmid depends on sigma 54-rna polymerase and requires the activator protein xylr that binds to the cis-acting upstream regulatory sequence of each promoter. for transcription of op1 in escherichia coli, integration host factor (ihf) is also required. ihf binds to dna between the upstream regulatory sequence and the promoter sequence of op1. we showed that ihf ...	1994	7896737
substrate specificity of catechol 2,3-dioxygenase encoded by tol plasmid pww0 of pseudomonas putida and its relationship to cell growth.	catechol 2,3-dioxygenase encoded by tol plasmid pww0 of pseudomonas putida consists of four identical subunits, each containing one ferrous ion. the enzyme catalyzes ring cleavage of catechol, 3-methylcatechol, and 4-methylcatechol but shows only weak activity toward 4-ethylcatechol. two mutants of catechol 2,3-dioxygenases (4ecr1 and 4ecr6) able to oxidize 4-ethylcatechol, one mutant (3mcs) which exhibits only weak activity toward 3-methylcatechol but retained the ability to cleave catechol and ...	1994	7928969
two identical copies of is1246, a 1275 base pair sequence related to other bacterial insertion sequences, enclose the xyl genes on tol plasmid pww0.	two identical direct repeats of a 1275 bp sequence, designated is1246, encompass the xyl genes, which determine the catabolism of toluene, m- and p-xylenes to central metabolites, on the tol catabolic plasmid pww0. is1246 has a terminal inverted repeat of 12 bp (5'gggcacctcgaa3') and contains a major open reading frame of 280 codons. this orf shows significant homology with orfs encoded by a number of bacterial insertion sequences from bacteroides, neisseria and escherichia coli.	1994	7952183
the organization of the pm promoter of the tol plasmid reflects the structure of its cognate activator protein xyls.	the toluate catabolic operon carried by the tol plasmid pww0 of pseudomonas putida is positively regulated by the benzoate-responsive xyls protein which, when activated, stimulates transcription from the operon promoter pm. in this study, the mode in which xyls effects the activity of the pm promoter was examined in vivo by genetic analysis of both protein and promoter variants. substitution of his31asp/ser32pro,leu113pro,phe214leu/glu215a sp/arg216pro or thr312pro, all predicted to disrupt the ...	1994	7969028
loss of the tol meta-cleavage pathway functions of pseudomonas putida strain paw1 (pww0) during growth on toluene.	a derivative of pseudomonas putida strain paw1 bearing the tol plasmid pww0 was isolated from a culture which has grown unlimited on toluene. in contrast to the parent strain paw1, the derivative, strain cg220, is unable to grow with xylenes and toluates, while toluene and benzoate served as substrates. strain cg220 had a remarkable growth advantage against the wild type when grown with toluene. biochemical analysis showed that in strain cg220 toluene was metabolised through the tol plasmid uppe ...	1994	7996396
codon usage patterns suggest independent evolution of two catabolic operons on toluene-degradative plasmid tol pww0 of pseudomonas putida.	tol plasmid pww0 of pseudomonas putida encodes a set of enzymes responsible for the degradation of toluene. the structural genes for these catobolic enzymes are clustered into two operons--namely, the xy/cmab and xy/xyzltegfjqkih operons. we examined the codon usage patterns of these catabolic genes by measuring the codon-usage distances between pairs of these catabolic genes. the codon-usage distance, d, between gene 1 and gene 2 was defined as d = [sigma(pj-qj)2]1/2, are the frequencies of the ...	1994	8007001
transposon-mediated mobilization of chromosomally located catabolic operons of the cam plasmid by tol plasmid transposon tn4652 and cam plasmid transposon tn3614.	the cam (camphor degradation) plasmid is integrated into the chromosome of pseudomonas putida paw-line strains and is not self-transferable as a plasmid via conjugation. our results show that the mobilization of chromosomally located cam and the integration of cam-operons into the chromosome of the new cam+ transconjugants is a reca-independent process mediated by transposons tn4652 (17 kbp) and tn3614 (7.2 kbp). transposon tn3614 is apparently identical to the left-hand and the right-hand seque ...	1994	8012608
functional and structural relationship of various extradiol aromatic ring-cleavage dioxygenases of pseudomonas origin.	the extradiol ring-cleavage dioxygenases derived from seven different pseudomonas strains were expressed in escherichia coli and the substrate specificities were investigated for a variety of catecholic compounds. the substrate range of four 2,3-dihydroxybiphenyl dioxygenases from biphenyl-utilizing bacteria, 3-methylcatechol dioxygenase from toluene utilizing pseudomonas putida f1, 1,2-dihydroxynaphthalene dioxygenase from a nah7 plasmid, and catechol 2,3-dioxygenase from a tol plasmid pww0 wer ...	1994	8020752
chromosomal gene capture mediated by the pseudomonas putida tol catabolic plasmid.	the pseudomonas putida tol plasmid pww0 is able to mediate chromosomal mobilization in the canonical unidirectional way, i.e., from donor to recipient cells, and bidirectionally, i.e., donor-->recipient-->donor (retrotransfer). transconjugants are recipient cells that have received dna from donor cells, whereas retrotransconjugants are donor bacteria that have received dna from a recipient. the tol plasmid pww0 is able to directly mobilize and retromobilize a kanamycin resistance marker integrat ...	1994	8045894
co-regulation by bent dna. functional substitutions of the integration host factor site at sigma 54-dependent promoter pu of the upper-tol operon by intrinsically curved sequences.	the role of integration host factor (ihf) in the regulation of the sigma 54-dependent promoter pu of the tol plasmid of pseudomonas putida has been examined. we have selected in vivo insertions of intrinsically curved dna that restore the responsiveness of an ihf-binding site deletion variant of pu to the cognate activator of the system, xylr. we found five pu derivatives which had inserted a core sequence with 6 phased [a]6 tracts, flanked by different lengths of dna at the location of the form ...	1994	8077217
tol plasmid-specified xylene oxygenase is a wide substrate range monooxygenase capable of olefin epoxidation.	xylene oxygenase, which is encoded on the tol plasmid pwwo of pseudomonas putida mt-2, is a key enzyme system in the degradation of toluene and xylenes by this organism. it was expressed in an escherichia coli recombinant strain carrying the xylma structural genes. this recombinant, which expressed xylene oxygenase from the heat-shock induced lambda pl promoter, was analyzed for its potential as a biocatalytic tool so as to effect the oxidation of side chains of aromatic hydrocarbons to the corr ...	1994	7764991
carbon source-dependent inhibition of xyl operon expression of the pseudomonas putida tol plasmid.	tol plasmid-encoded degradation of benzyl alcohol by pseudomonas putida is inhibited by glucose and other compounds related to the main carbohydrate metabolism in pseudomonas species. we report here that this effect is exerted at the level of expression of the xyl catabolic operons, and two xyl promoters, pu and ps, were identified as the primary targets of this inhibition. xyl promoter activation was also inhibited by glucose in the heterologous escherichia coli system, apparently not however b ...	1994	8132475
genetic evidence for activation of the positive transcriptional regulator xy1r, a member of the ntrc family of regulators, by effector binding.	the xy1r protein positively controls expression from the pseudomonas putida tol plasmid sigma 54-dependent pu and ps promoters, in response to the presence of aromatic effectors such as m-xylene, m-methylbenzyl alcohol, and p-chlorobenzaldehyde in the culture medium. xy1r also autoregulates its own synthesis. a mutant xy1r regulator called xy1r7 was isolated after nitrosoguanidine mutagenesis of the wild-type gene and phenotypic selection for mutants that had acquired the ability to recognize m- ...	1994	8132529
transcriptional induction kinetics from the promoters of the catabolic pathways of tol plasmid pww0 of pseudomonas putida for metabolism of aromatics.	we determined, under several growth conditions, the kinetics of mrna synthesis from the four pseudomonas putida pww0 plasmid promoters involved in degradation of xylenes and methylbenzyl alcohols via toluates. transcription by xyls of the meta-cleavage pathway operon promoter (pm) for the metabolism of alkylbenzoates was stimulated immediately after the addition of an effector, both in luria-bertani (lb) medium and in minimal medium. activation of the sigma 54-dependent upper-pathway operon prom ...	1994	8169200
genetic evidence that the xyls regulator of the pseudomonas tol meta operon controls the pm promoter through weak dna-protein interactions.	the activation of the pm promoter of the meta operon of the tol plasmid of pseudomonas putida by its cognate xyls activator protein in the presence and absence of benzoate inducers has been examined in specialized escherichia coli strains carrying pm-lacz fusions and the xyls gene in different configurations in which all controlling elements are present in near native conditions and stoichometry. expression of a chromosomal pm-xylx::lacz fusion was primarily dependent on the addition of an effec ...	1994	8195070
metabolic engineering of pseudomonas putida for the simultaneous biodegradation of benzene, toluene, and p-xylene mixture.	for the complete biodegradation of a mixture of benzene, toluene, and p-xylene (btx), a critical metabolic step that can connect two existing metabolic pathways of aromatic compounds (the tod and the tol pathways) was determined. toluate-cis-glycol dehydrogenase in the tol pathway was found to attack benzene-cis-glycol, toluene-cis-glycol, and p-xylene-cis-glycol, which are metabolic intermediates of the tod pathway. based on this observation, a hybrid strain, pseudomonase putida tb101, was cons ...	1994	18615528
fate of pseudomonas putida after release into lake water mesocosms: different survival mechanisms in response to environmental conditions.	to study the fate of pseudomonas putida dsm 3931 in an aquatic environment, cultures of the strain were released into lake water mesocosms. p. putida, bearing the tol-plasmid, was released as a representative xenobiotic-degrading microorganism. the release was carried out in mesocosms with unamended lake water and in lake water with added culture medium to compare the survival of the strain due to the influence of different organic load. as a comparison, the survival of p. putida was followed in ...	1994	24190270
analysis of the mrna structure of the pseudomonas putida tol meta fission pathway operon around the transcription initiation point, the xylte and the xylfj regions.	the 13 genes encoded by the meta-cleavage operon (approx. 11 kb) of pseudomonas putida tol plasmid pww0 are transcribed from a single promoter, pm. in p. putida, transcription from pm was strictly dependent on the presence of effector-activated xyls protein. three regions of the transcript were analyzed in the wild-type strain of p. putida (pww0) by s1 nuclease protection and primer extension analyses. a major point of transcription initiation was found in the most 5'-end of the operon, which de ...	1993	8241263
the specific growth rate of pseudomonas putida paw1 influences the conjugal transfer rate of the tol plasmid.	the kinetics of the conjugal transfer of a tol plasmid were investigated by using pseudomonas putida paw1 as the donor strain and p. aeruginosa pao 1162 as the recipient strain. short-term batch mating experiments were performed in a nonselective medium, while the evolution of the different cell types was determined by selective plating techniques. the experimental data were analyzed by using a mass action model that describes plasmid transfer kinetics. this method allowed analysis of the mating ...	1993	8250565
new derivatives of tol plasmid pww0.	two new segregants, ppw1-1 and ppw161-1, of pseudomonas putida were isolated from the stock cultures paw85(pww0) and paw85(pww0-161). strain ppw1-1 had lost its ability to grow on m-xylene but was able to grow on m-toluate. a deletion of the left-hand of transposon tn4651, including the upper-operon genes, had taken place in plasmid pww0mut1, isolated from strain ppw1-1. additional deletions were observed in pww0mut1 after 'benzoate-curing' (plasmids pww0mut15, pww0mut19, pww0mut27). the genes o ...	1993	8254307
a substrate-dependent biological containment system for pseudomonas putida based on the escherichia coli gef gene.	a model substrate-dependent suicide system to biologically contain pseudomonas putida kt2440 is reported. the system consists of two elements. one element carries a fusion between a synthetic lac promoter (pa1-04/03) and the gef gene, which encodes a killing function. this element is contained within a transposaseless mini-tn5 transposon so that it can be integrated at random locations on the pseudomonas chromosome. the second element, harbored by plasmid pcc102, is designed to control the first ...	1993	8285679
in vivo reactivation of catechol 2,3-dioxygenase mediated by a chloroplast-type ferredoxin: a bacterial strategy to expand the substrate specificity of aromatic degradative pathways.	the meta-cleavage operon of the tol plasmid pww0 of pseudomonas putida contains 13 genes responsible for the oxidation of benzoate and toluates to krebs cycle intermediates via estradiol (meta) cleavage of (methyl)catechol. the functions of all the genes are known with the exception of xylt. we constructed pww0 mutants defective in the xylt gene, and found that these mutants were not able to grow on p-toluate while they were still capable of growing on benzoate and m-toluate. in the xylt mutants ...	1993	8344270
analysis of pseudomonas gene products using laciq/ptrp-lac plasmids and transposons that confer conditional phenotypes.	novel transposon and plasmid-based broad-host-range expression systems have been developed to facilitate the genetic analysis of gene products of pseudomonas and related gram- bacteria. the properties of laciq/ptrp-lac were used to construct mini-tn5 expression vector transposons and rsf1010-derived plasmids for controlled expression and generation of conditional phenotypes. these plasmids were used to hyper-express the xyls regulator of the meta operon of the tol plasmid of p. putida or the bph ...	1993	8380783
construction of a pseudomonas hybrid strain that mineralizes 2,4,6-trinitrotoluene.	a bacterium, pseudomonas sp. strain c1s1, able to grow on 2,4,6-trinitrotoluene (tnt), 2,4- and 2,6-dinitrotoluene, and 2-nitrotoluene as n sources, was isolated. the bacterium grew at 30 degrees c with fructose as a c source and accumulated nitrite. through batch culture enrichment, we isolated a derivative strain, called pseudomonas sp. clone a, which grew faster on tnt and did not accumulate nitrite in the culture medium. use of tnt by these two strains as an n source involved the successive ...	1993	8468288
a comparison of the multiple alleles of xyls carried by tol plasmids pww53 and pdk1 and its implications for their evolutionary relationship.	both of the independently isolated tol plasmids pww53 and pdk1 contain multiple regions homologous to the xyls regulatory gene of the archetypal tol plasmid pww0. the three homologues on pww53 vary in the extent of their homology to xylspww0, xyls1pww53 is 99% identical to xylspww0 and is located relative to the single copy of xylrpww53 in exactly the same way as xyls and xylr on pww0. the dna sequence of xyls3pww53 is 87% identical to the xylspww0 sequence within the coding region but the non-c ...	1993	8473862
identification of a cis-acting sequence within the pm promoter of the tol plasmid which confers xyls-mediated responsiveness to substituted benzoates.	the dna sequences within the pm promoter/operator region of the meta operon of the tol plasmid of pseudomonas putida, which confer xyls-mediated responsiveness to substituted benzoates, have been identified through deletion analysis and mutagenesis of the region. integrity and proper phasing of two homologous tandem sequences 5'-tgcaapuaapu-pyggnta-3', separated by six base-pairs and overlapping with the -35 region of the pm promoter, was essential for m-toluate activation of a pm-lacz fusion in ...	1993	8478926
kinetic studies on benzyl alcohol dehydrogenase encoded by tol plasmid pwwo. a member of the zinc-containing long chain alcohol dehydrogenase family.	the nucleotide sequence of the structural gene for benzyl alcohol dehydrogenase encoded by tol plasmid pwwo of pseudomonas putida has been determined. benzyl alcohol dehydrogenase is a member of the long-chain zinc alcohol dehydrogenase family and, like other alcohol dehydrogenases of this family, contains two zinc atoms per subunit. benzyl alcohol dehydrogenase, while sharing 31% identical residues with horse liver alcohol dehydrogenase, contains several amino acid substitutions near the active ...	1993	8496150
physical organization of the upper pathway operon promoter of the pseudomonas tol plasmid. sequence and positional requirements for xylr-dependent activation of transcription.	the upper pathway operon of the pseudomonas putida tol plasmid belongs to the -12/-24 class of promoters. these promoters exhibit three regions critical for regulated transcription, namely, the -12/-24 site for rna polymerase/sigma 54 binding, the -55/-67 region for ihf protein binding, and the -130(uas2)/-170(uas1) region, where two sites for xylr binding are located. the xylr-protected g residues located at -131, -139, -160 and -169 were replaced with as, and the activity of the mutant promote ...	1993	8510657
comparison of the nucleotide sequences of the meta-cleavage pathway genes of tol plasmid pww0 from pseudomonas putida with other meta-cleavage genes suggests that both single and multiple nucleotide substitutions contribute to enzyme evolution.	tol plasmid pww0 from pseudomonas putida mt-2 encodes catabolic enzymes required for the oxidation of toluene and xylenes. the structural genes for these catabolic enzymes are clustered into two operons, the xylcmabn operon, which encodes a set of enzymes required for the transformation of toluene/xylenes to benzoate/toluates, and the xylxyzltegfjqkih operon, which encodes a set of enzymes required for the transformation of benzoate/toluates to krebs cycle intermediates. the latter operon can be ...	1993	8510667
mineralization of p-methyl-14c-benzoate in soils by pseudomonas putida (pww0).	pseudomonas putida bearing the archetypal tol plasmid pww0 metabolizes p-methylbenzoate through a meta-cleavage pathway. in complex environments such as soils that are relatively rich in organic matter, we observed metabolism of p-methyl-14c-benzoate, which could be monitored as 14co2 evolution from the labelled alkylaromatic. linear 14co2 evolution in soils took place for at least a month, although efficient mineralization of the alkylaromatic required appropriate mass transfer to allow the bac ...	1993	8111534
transcriptional control of the pseudomonas putida tol plasmid catabolic pathways.	tol plasmid pww0 of pseudomonas putida contains two operons that specify a pathway for the degradation of aromatic hydrocarbons. the upper pathway operon encodes the enzymes for the oxidation of toluene/xylenes to benzoate/toluates, and the metacleavage pathway operon encodes the enzymes for the further oxidation of these compounds to krebs cycle intermediates. their expression is controlled by the gene products of two divergently transcribed regulatory genes, xyir and xyis. the xyir protein, wh ...	1993	7934920
identification of critical amino-terminal regions of xyls. the positive regulator encoded by the tol plasmid.	the xyls protein is the positive regulator of the promoter controlling the meta-cleavage pathway (pm) for catabolism of certain alkylbenzoates on the tol plasmid of pseudomonas. transcription from pm is mediated by xyls either in the presence of benzoate effectors or through xyls hyperproduction. two regions of the nh2 terminus of xyls (residues 37-45) had been predicted to be involved in effector control of xyls transcriptional activation. different methods were used to induce mutations in this ...	1992	1429638
expression and transfer of engineered catabolic pathways harbored by pseudomonas spp. introduced into activated sludge microcosms.	two genetically engineered microorganisms (gems), pseudomonas sp. strain b13 fr1(pfrc20p) (fr120) and pseudomonas putida kt2440(pwwo-eb62) (eb62), were introduced into activated sludge microcosms that had the level of aeration, nutrient makeup, and microbial community structure of activated sludge reactors. fr120 contains an experimentally assembled ortho cleavage route for simultaneous degradation of 3-chlorobenzoate (3cb) and 4-methyl benzoate (4mb); eb62 contains a derivative tol plasmid-enco ...	1992	1444370
physiological properties of a pseudomonas strain which grows with p-xylene in a two-phase (organic-aqueous) medium.	pseudomonas putida idaho utilizes toluene, m-xylene, p-xylene, 1,2,4-trimethylbenzene, and 3-ethyltoluene as growth substrates when these hydrocarbons are provided in a two-phase system at 5 to 50% (vol/vol). growth also occurs on luria-bertani medium in the presence of a wide range of organic solvents. the ability of the organism to grow in the presence of organic solvents is correlated with the logarithm of the octanol-water partition coefficient, with dimethyl-phthalate (log p(oct) = 2.3) bei ...	1992	1444381
construction of a broad host range shuttle vector for gene cloning and expression in actinobacillus pleuropneumoniae and other pasteurellaceae.	we have constructed a pair of broad host range expression vectors, pjff224-nx and pjff224-xn, based on plasmid rsf1010, which enable cloning and efficient expression of genes in actinobacillus pleuropneumoniae and pasteurella haemolytica and in escherichia coli. the vectors consist of the minimal autonomous replicon of the broad host range plasmid rsf1010 and a type ii chloramphenicol acetyl transferase gene for chloramphenicol resistance selection. in addition, they contain a gene expression ca ...	1992	1448612