Showing papers on "Pseudomonas putida published in 1987"
TL;DR: A series of derivatives of the omega interposon that can be used for in vitro insertional mutagenesis are constructed and insertion of these interposons in the plasmid between the promoter and the catechol 2,3-dioxygenase (C23O) gene dramatically reduced the expression of this enzyme in Escherichia coli.
714 citations
TL;DR: Inoculation of canola seeds with a nitrogen-fixing strain of Pseudomonas putida drastically increased the root length of seedlings grown in sterile growth pouches and added phosphate to the growth solution at similar concentrations stimulated root elongation of inoculated and noninoculated seedlings.
Abstract: Inoculation of canola (Brassica campestris) seeds with a nitrogen-fixing strain of Pseudomonas putida (GR12-2) drastically increased the root length of seedlings grown in sterile growth pouches. Se...
351 citations
TL;DR: Three critical enzymes catechol oxygenase II (chlorocatechol dioxygenase), muconate cycloisomerase II, and dienelactone hydrolase, are involved in the degradation of chlorocatechols, which are obligatory intermediates in the catabolism of chlorinated aromatic compounds.
Abstract: Three critical enzymes catechol oxygenase II (chlorocatechol dioxygenase), muconate cycloisomerase II, and dienelactone hydrolase, are involved in the degradation of chlorocatechols, which are obligatory intermediates in the catabolism of chlorinated aromatic compounds. The organization and complete nucleotide sequence of the genes for these enzymes have been determined on a 4.2-kilobase-pair (kbp) Bgl II fragment cloned from the plasmid pAC27, based on the agreement of open reading frame lengths with apparent mobilities of polypeptides expressed in Escherichia coli maxicells, predicted N-terminal amino acid sequences with those of the purified proteins, and predicted total amino acid compositions with those of the purified proteins. The 4.2-kbp fragment contains the three genes and ribosome binding sites for those genes but no promoter. When placed downstream of the tac promoter in the broad-host-range plasmid pMMB22, this fragment directs the synthesis of all three enzymes in both E. coli and Pseudomonas putida only on induction with isopropyl beta-D-thiogalactopyranoside, suggesting that the gene cluster is regulated as a single unit under the control of a single promoter. Endogenous transcription initiation of the gene cluster on pAC27, however, occurs from a site present within a 386-bp Bgl II fragment upstream of the 4.2-kbp fragment, and sequences 5' to that site are similar to the sequences of other positively controlled Pseudomonas promoters occurring on the TOL and NAH plasmids.
187 citations
TL;DR: Electron microscopy showed that both strains of Pseudomonas putida and P. fluorescens were enrobed in fibrous glycocalyces and that these structures were involved in attachment of the cells to a solid surface and as structural matrices in the microcolony mode of growth.
Abstract: In this study, the adhesive exopolysaccharides of strains of Pseudomonas putida and P. fluorescens, both isolated from freshwater epilithic communities, were examined with regard to their chemical ...
138 citations
TL;DR: The monooxygenase cytochrome P-450cam from the soil bacterium Pseudomonas putida is shown to catalyze the hydroxylation of the substrate analogue norcamphor (I) to form 5- (II), 6- (III), and 3-exo-hydroxynorcamphor(IV) as discussed by the authors.
Abstract: The monooxygenase cytochrome P-450cam from the soil bacterium Pseudomonas putida is shown to catalyze the hydroxylation of the substrate analogue norcamphor (I) to form 5- (II), 6- (III), and 3-exo-hydroxynorcamphor (IV).
118 citations
TL;DR: Alk expression is very efficient and strictly regulated to both P. putida and E. coli, and could be further reduced to a 5-kilobase pair operon without affecting the Alk phenotype in either species to a significant extent.
113 citations
TL;DR: Comparison of the nucleotide and amino acid sequences for catB with the corresponding sequences of the clcB gene showed significant homology, suggesting that catB andClcB have diverged from a common ancestral gene.
112 citations
TL;DR: The nucleotide sequence of the genes from Pseudomonas putida encoding oxidation of benzene to catechol was determined and the relation between the product of each cistron and the components of the benzene oxidation enzyme system is discussed.
Abstract: The nucleotide sequence of the genes from Pseudomonas putida encoding oxidation of benzene to catechol was determined. Five open reading frames were found in the sequence. Four corresponding protein molecules were detected by a DNA-directed in vitro translation system. Escherichia coli cells containing the fragment with the four open reading frames transformed benzene to cis-benzene glycol, which is an intermediate of the oxidation of benzene to catechol. The relation between the product of each cistron and the components of the benzene oxidation enzyme system is discussed.
111 citations
TL;DR: It is postulated that failure of siderophore-negative Tn5 transposon mutants of isolate WCS358 lo induce growth stimulation demonstrates that sidersophore production is a prerequisite for growth stimulation in short potato-rotation soil.
Abstract: Summary-A bioassay is described for studying the mechanisms of growth stimulation by fluorescent pseudomonads using rooted potato stem cuttings. Root development of potato stem cuttings was inhibited in short potato-rotation soil compared to development in long potato-rotation soil. Treatment with Pseudomonas Juorescens isolate WCS374 or Pseudomonas putida isolate WCS358 increased root development in short potato-rotation soil, whereas siderophore-negative Tn5 transposon mutants of isolate WCS358 had no effect. Both siderophore-producing and siderophore-negative Tn5 mutants of isolate WCS358 could be recovered in similar numbers from the root systems. These results were obtained in a bioassay of 8 days duration. It is postulated that failure of siderophore-negative Tn5 transposon mutants of isolate WCS358 lo induce growth stimulation demonstrates that siderophore production is a prerequisite for growth stimulation in short potato-rotation soil.
92 citations
TL;DR: Storage experiments revealed an excellent longterm storage capability of the biocatalyst: activated carbon with adsorbed cells of Pseudomonas putida P8 and Cryptococcus elinovii H1 could be stored up to 12 months without decrease on degradation capacity.
Abstract: Phenol degradation by a defined mixed culture of Pseudomonas putida P8 and Cryptococcus elinovii H 1, which were immobilized by adsorption on activated carbon, was studied. The immobilized mixed culture was able to degrade phenol up to 17 g/l and degraded it faster than the pure cultures, depending on a “complementary metabolism” of the two microorganisms. Storage experiments revealed an excellent longterm storage capability of the biocatalyst: activated carbon with adsorbed cells of Pseudomonas putida P8 and Cryptococcus elinovii H1 could be stored up to 12 months without decrease on degradation capacity. Scanning electron micrographs showed that Pseudomonas putida P8 had grown through the pore system of the activated carbon into the inside of the carbon particles.
90 citations
TL;DR: A set of broad-host-range vectors allowing direct selection of recombinant DNA molecules to facilitate subcloning and expression analyses of Pseudomonas genes was constructed using Bg/II lacZ alpha cassette and Controlled expression vectors pVDtac39 and pVDTac24 were shown to be useful for determination of enzymatic activities encoded by the cloned DNA fragments and determination of the corresponding polypeptides.
TL;DR: Two separate continuous cultures of Pseudomonas putida R5-3 and CB1-9 were concentrated and continuously amalgamated on a ceramic bead column, which was subjected to a continuous stream of chlorobenzene vapors, which indicated rearrangement of plasmid DNA.
Abstract: Separate continuous cultures of Pseudomonas putida R5-3, grown on toluene, and Pseudomonas alcaligenes C-O, grown on benzoate, were concentrated and continuously amalgamated on a ceramic bead column, which was subjected to a continuous stream of chlorobenzene vapors. A recombinant strain, P. putida CB1-9, was isolated in less than 1 month. P. alcaligenes C-0 grew on benzoate and 3-chlorobenzoate but not on toluene, P. putida R5-3 grew on benzoate and toluene but not on 3-chlorobenzoate, and neither strain grew on chlorobenzene or 1,4-dichlorobenzene; however, the recombinant P. putida CB1-9 grew on all of these substrates. Chlorobenzene-utilizing strains were not found in continuous cultures run at the lowest growth rate (0.05/h) or in the absence of the donor strain, P. alcaligenes C-0. Chloride was released in stoichiometric amounts when P. putida CB1-9 was grown on either chlorobenzene or 1,4-dichlorobenzene. The recombinant strain was related to P. putida R5-3, phenotypically and genetically. Restriction enzyme digests of the single 57-kilobase (kb) plasmid in R5-3 and of the single 33-kb plasmid in CB1-9 were similar, but also indicated rearrangement of plasmid DNA. Coincidental or causal to the loss of the 24-kb fragment was the observation that the recombinant--unlike its parent, R5-3--did not grow on xylenes or methylbenzoates. Although both ortho-pyrocatechase (OP) and meta-pyrocatechase (MP) were found in CB1-9 and R5-3, MP activity was 20- to 50-fold higher in R5-3 cells grown on 4-methylbenzoate than in the same cells grown on benzene.(ABSTRACT TRUNCATED AT 250 WORDS) Images
TL;DR: This chapter describes the assay method, purification procedures, and general properties of L-methionine γ -lyase from Pseudomonas putida and Aeromonas and its role in α-ketobutyrate, methanethiol, and ammonia production.
Abstract: Publisher Summary This chapter describes the assay method, purification procedures, and general properties of L-methionine γ -lyase from Pseudomonas putida (P. putida) and Aeromonas. L-Methionine γ-lyase catalyzes α, γ-elimination of L- methionine to produce α-ketobutyrate, methanethiol, and ammonia. The enzyme has been purified from Clostridium sporogenes, Pseudomonas putida and Aeromonas. The enzymatic α, γ-elimination of L-methionine can be routinely followed by spectrophotometric determination of α-ketobutyrate with 3-methyl-2-benzothiazolone hydrazone hydrochloride. In the case of preparation from P. putida, Pseudomonas putida ICR 3460 is grown in a medium containing 0.25% L-methionine, 0.1% polypeptone, 0.1% glycerol, 0.1% KH2PO4, 0.1% K2HPO4, 0.01% MgSO4·7H2O, and 0.025% yeast extract at 28° for 18 hr with shaking. The enzyme is crystallized by the vapor diffusion method of McPherson. L-Methionine γ-lyase is also purified from Aeromonas sp. ICR 3470 essentially by the same method as described for preparation from P. putida, using DEAE-Toyopearl 650 M and DE AE- Sephadex A-50 column chromatography. About 30 mg of a homogeneous preparation (specific activity, 31 units/mg) is obtained from a crude extract of 204 g of wet cells (total protein, 4.6 g; specific activity, 1.04 units/rag), with an overall yield of 20%.
TL;DR: The equation governing the instant uptake of phenol by microorganisms in chemostat in the high conversion range of Phenol can be used to determine the stability range of continuous stirred tank biochemical reactors (CSTBR) utilizing phenol.
Abstract: The specific uptake rate of phenol by washed cells of Pseudomonas putida grown on phenol in steady-state continuous culture at various dilution rates was studied. The Monod-Haldane-type equation was applied to fit the data and the best kinetic parameters were determined by nonlinear least-squares techniques. The values of the kinetic parameters were found to increase monotonically with the phenol concentration in the original chemostat. The relations between the values of kinetic parameters and phenol concentration in the chemostat were described by empirical equations. Then the equation governing the instant uptake of phenol by microorganisms in chemostat in the high conversion range of phenol was proposed. This equation together with the mass balance equations can be used to determine the stability range of continuous stirred tank biochemical reactors (CSTBR) utilizing phenol.
TL;DR: The complete nucleotide sequence of the clcD gene and the amino acid sequence of dienelactone hydrolase deduced from the DNA sequence are presented and an apparently conserved pentapeptide neighboring a cysteinyl side chain that presumably lies at or near the active sites is suggested.
Abstract: The clcD structural gene encodes dienelactone hydrolase (EC 3.1.1.45), an enzyme that catalyzes the conversion of dienelactones to maleylacetate. The gene is part of the clc gene cluster involved in the utilization of chlorocatechol and is carried on a 4.3-kilobase-pair BglII fragment subcloned from the Pseudomonas degradative plasmid pAC27. A 1.9-kilobase-pair PstI-EcoRI segment subcloned from the BglII fragment was shown to carry the clcD gene, which was expressed inducibly under the tac promoter at levels similar to those found in 3-chlorobenzoate-grown Pseudomonas cells carrying the plasmid pAC27. In this study, we present the complete nucleotide sequence of the clcD gene and the amino acid sequence of dienelactone hydrolase deduced from the DNA sequence. The NH2-terminal amino acid sequence encoded by the clcD gene from plasmid pAC27 corresponds to a 33-residue sequence established for dienelactone hydrolase encoded by the Pseudomonas sp. strain B13 plasmid pWR1. A possible relationship between the clcD gene and pcaD, a Pseudomonas putida chromosomal gene encoding enol-lactone hydrolase (EC 3.1.1.24) is suggested by the fact that the gene products contain an apparently conserved pentapeptide neighboring a cysteinyl side chain that presumably lies at or near the active sites; the cysteinyl residue occupies position 60 in the predicted amino acid sequence of dienelactone hydrolase.
TL;DR: Two clones conferring upon Agrobacterium the ability to catabolize the mannityl opines were mobilized to several Rhizobium sp.
Abstract: The genetic organization of functions responsible for mannityl opine catabolism of the Ti plasmid of Agrobacterium tumefaciens strain 1,5955 was investigated. A partial HindIII digest of pTi1,5955 was cloned into a broad host range cosmid and the clones obtained were tested for ability to confer mannityl opine degradation upon Agrobacterium. Inserts containing genes for catabolism of mannopinic acid, mannopine, agropine, and agropinic acid were obtained, spanning a segment of 43 kb on the Ti plasmid. Two clones conferring upon Agrobacterium the ability to catabolize the mannityl opines were mobilized to several Rhizobium sp., to Pseudomonas putida and P. fluorescens and to Escherichia coli. The catabolic functions were phenotypically expressed in all Rhizobium sp. tested, and in P. fluorescens, but not in P. putida or in E. coli.
TL;DR: A phyllospheric bacterial culture found to contain a fluorescent pseudomonas which was identified as Pseudomonas putida and a Corynebacterium sp.
Abstract: A phyllospheric bacterial culture, previously reported to partially replace nitrogen fertilizer (B. R. Patti and A. K. Chandra, Plant Soil 61:419-427, 1981) was found to contain a fluorescent pseudomonas which was identified as Pseudomonas putida and a Corynebacterium sp. The P. putida isolate was found to produce an extracellular cutinase when grown in a medium containing cutin, the polyester structural component of plant cuticle. The Corynebacterium sp. grew on nitrogen-free medium but could not produce cutinase under any induction conditions tested, whereas P. putida could not grow on nitrogen-free medium. When cocultured with the nitrogen-fixing Corynebacterium sp., the P. putida isolate grew in a nitrogen-free medium, suggesting that the former provided fixed N2 for the latter. These results suggest that the two species coexist on the plant surface, with one providing carbon and the other providing reduced nitrogen for their growth. The presence of cutin in the medium induced cutinase production by P. putida. However, unlike the previously studied fungal systems, cutin hydrolysate did not induce cutinase. Thin-layer chromatographic analysis of the products released from labeled apple fruit cutin showed that the extracellular enzyme released all classes of cutin monomers. This enzyme also catalyzed hydrolysis of the model ester substrates, p-nitrophenyl esters of fatty acids, and optimal conditions were determined for a spectrophotometric assay with p-nitrophenyl butyrate as the substrate. It did not hydrolyze triacyl glycerols, indicating that the cutinase activity was not due to a nonspecific lipase. It showed a broad pH optimum between 8.0 and 10.5 with 3H-labeled apple cutin as the substrate.(ABSTRACT TRUNCATED AT 250 WORDS)
TL;DR: Metabolism of the aza-arenes quinoline, isoquinoline, quinazoline, and quinoxaline by a mutant strain of the bacterium Pseudomonas putida resulted in attack at the carbocyclic ring (to yield stable cis-dihydrodiols and phenols) and at the heterocycling ring ( to yield phenols and ring cleavage products).
Abstract: Metabolism of the aza-arenes quinoline, isoquinoline, quinazoline, and quinoxaline by a mutant strain of the bacterium Pseudomonas putida resulted in attack at the carbocyclic ring (to yield stable cis-dihydrodiols and phenols) and at the heterocyclic ring (to yield phenols and ring cleavage products).
TL;DR: Increased activities of peroxidase and indole 3-acetic acid (IAA) oxidase were detected on root surfaces of bean (Phaseolus vulgaris) seedlings colonized with a soil saprophytic bacterium, Pseudomonas putida.
Abstract: Increased activities of peroxidase and indole 3-acetic acid (IAA) oxidase were detected on root surfaces of bean (Phaseolus vulgaris) seedlings colonized with a soil saprophytic bacterium, Pseudomonas putida. IAA oxidase activity increased over 250-fold and peroxidase 8-fold. Enhancement was greater for 6-day-old than for 4- or 8-day-old inoculated plants No IAA oxidase or peroxidase activities were associated with the bacterial cells. Native polyacrylamide gel electrophoresis demonstrated that washes of P. putida-inoculated roots contained two zones of peroxidase activity. Only the more anodic bands were detected in washes from noninoculated roots. Ion exchange and molecular sizing gel chromatography of washes from P. putida-colonized roots separated two fractions of peroxidase activity. One fraction corresponded to the anodic bands detected in washes of P. putida inoculated and in noninoculated roots. A second fraction corresponded to the less anodic zone of peroxidase, which was characteristic of P. putida-inoculated plants. This peroxidase had a higher IAA oxidase to peroxidase ratio than the more anodic, common enzyme. The changes in root surface peroxidases caused by colonization by a saprophytic bacterium are discussed with reference to plant-pathogen interactions.
TL;DR: Amino acid sequence studies show that the aliphatic amidase from Pseudomonas aeruginosa PAC142 consists of a single polypeptide chain of 346 residues, giving an M r of 38400.
TL;DR: The terminal oxygenase component of benzene dioxygenase from Pseudomonas putida strain ML2 was shown to contain two subunits, of Mr 54,500 and 23,500, by SDS/polyacrylamide-gel electrophoresis, and polypeptides were present only when this strain was grown on toluene.
Abstract: The terminal oxygenase component of benzene dioxygenase from Pseudomonas putida strain ML2 was shown to contain two subunits, of Mr 54,500 and 23,500, by SDS/polyacrylamide-gel electrophoresis. The native Mr of the terminal oxygenase was estimated to be 168,000 +/- 4000. Polyclonal antibodies raised against each of the subunits cross-reacted with two polypeptides in cell-free extracts from toluene-grown Pseudomonas putida strain N.C.I.B. 11767. The Mr values of these polypeptides were similar to those reported for the subunits from the terminal dioxygenase component of toluene dioxygenase. These polypeptides were present only when this strain was grown on toluene. No cross-reactivity was observed with subunits of the naphthalene dioxygenase or benzoate dioxygenase systems.
TL;DR: The structural genes of the Pseudomonas oleovorans alk (alkane utilization) system, which are localized on the alkBAC operon, were cloned as a 16.9-kilobase pair EcoRI fragment and six different proteins were identified.
TL;DR: An autochthonous microflora developed on the carbon surface which could be demonstrated by scanning electron microscopy and culturing methods and reduced E. coli, P. aeruginosa, and P. putida densities in the effluent by a factor of more than 10(5) within 1 to 5 days.
Abstract: The survival of selected hygienically relevant bacterial species in activated carbon (AC) filters on a bench scale was investigated. The results revealed that after inoculation of the test strains the previously sterilized AC absorbed all bacteria (10(6) to 10(7)). After a period of 6 to 13 days without countable bacteria in the effluent, the numbers of Escherichia coli, Pseudomonas aeruginosa, and Pseudomonas putida increased up to 10(4) to 10(5) CFU/ml of effluent and 10(6) to 10(7) CFU/g of AC. When Klebsiella pneumoniae and Streptococcus faecalis were used, no growth in filters could be observed. The numbers of E. coli, P. aeruginosa, and P. putida, however, decreased immediately and showed no regrowth in nonsterile AC from a filter which had been continuously connected to running tap water for 2 months. Under these conditions an autochthonous microflora developed on the carbon surface which could be demonstrated by scanning electron microscopy and culturing methods (heterotrophic plate count). These bacteria reduced E. coli, P. aeruginosa, and P. putida densities in the effluent by a factor of more than 10(5) within 1 to 5 days. The hypothesis that antagonistic substances of the autochthonous microflora were responsible for the elimination of the artificial contamination could not be confirmed because less than 1% of the isolates of the autochthonous microflora were able to produce such substances as indicated by in vitro tests. Competition for limiting nutrients was thought to be the reason for the observed effects.
TL;DR: 3,4, 4,5, and 3,5-Difluoro-3-5-cyclohexadiene- cis -1,2-diol-1-carboxylates were prepared by microbial oxidation with a mutant strain of Pseudomonas putida JT 103.
TL;DR: A direct selection system for growth on valine-isoleucine agar was achieved by complementation of P. putida branched-chain keto acid dehydrogenase mutants by cloning into the broad-host-range vector pKT230.
Abstract: We cloned the structural genes for the individual subunits of the branched-chain keto acid dehydrogenase multienzyme complex on a 78-kilobase EcoRI-SstI restriction fragment of Pseudomonas putida chromosomal DNA by cloning into the broad-host-range vector pKT230 A direct selection system for growth on valine-isoleucine agar was achieved by complementation of P putida branched-chain keto acid dehydrogenase mutants The recombinant plasmid, pSS1-1, increased expression of branched-chain keto acid dehydrogenase up to five times in wild-type P putida The complex was expressed constitutively in P putida(pSS1-1) but was inducible in Escherichia coli HB101(pSS1-1) by high valine E coli minicells transformed with pSS1-1 produced three polypeptides which did not match the four polypeptides of the purified complex To resolve this problem, we inserted P putida DNA from pSS1-1 into pUC18 and pUC19 The pUC-derived plasmids were used as DNA templates in an E coli transcription-translation system Four polypeptides were produced from the pUC18-derived plasmid which had the correct molecular weights, showing that the structural genes had been cloned Since only weak bands were produced with the pUC19-derived plasmid, the direction of transcription was established The locations and order of all the structural genes of branched-chain keto acid dehydrogenase were located by restriction enzyme mapping Images
TL;DR: A bacterium capable of growth on 4-ethylphenol was isolated from soil and identified as Pseudomonas putida, and the enzymes of the proposed pathway were induced by growth on 5- methylphenol or 4-hydroxyacetophenone.
Abstract: SUMMARY: A bacterium capable of growth on 4-ethylphenol was isolated from soil and identified as Pseudomonas putida. Intact cells grown on 4-ethylphenol rapidly oxidized 4-hydroxyaceto-phenone as well as growth substrate and the bacterium was also capable of growth on 4-hydroxyacetophenone. The initial enzymes for 4-ethylphenol catabolism were still present, although at lower activities, in succinate-grown cells which oxidized 4-ethylphenol to 4-hydroxyacetophenone. Extracts of 4-ethylphenol-grown cells oxidized 4-hydroxyacetophenone when provided with NADPH. When this activity was partially purified a stoichiometry of 1 μmol O2 consumed per μmol of substrate was observed with the production of hydroquinone as required for a monooxygenase producing 4-hydroxyphernyl acetate followed by hydrolysis by an esterase. Cell extracts contained esterase activity and hydrolysed 4-hydroxyphenyl acetate to yield hydroquinone. Intact cells converted the analogue, acetophenone, into phenol. Hydroquinone served as the ring-fission substrate and was cleaved by an O2-requiring reaction. The enzymes of the proposed pathway were induced by growth on 4-ethylphenol or 4-hydroxyacetophenone.
TL;DR: The results suggest that TCG is the mediate of a gradual deterioration in the state of the culture which leads to a loss of both in vivo and in vitro toluence dioxygenase activity and a marked decrease in culture viability.
Abstract: Toluene was oxidized by a mutant strain of Pseudomonas putida (strain NG1) to toluene cis-glycol (TCG). Product was accumulated in fed-batch cultures to concentrations (18-24 g/L) higher than hitherto achieved. In vitro activities of toluene dioxygenase from P. putida NG1 were fivefold lower than that from the toluene-grown wild-type organism, whereas comparable activities of both catechol 2,3- and catechol 1,2-oxygenase were obtained; irreversible inhibition of toluene dioxygenase activity by TCG was shown in vitro. Ammonia deprivation during the production phase limited the growth of revertant organisms but had little effect on either the duration (25 h) of the process or the final concentration of TCG achieved. The rates of glucose utilization decreased throughout the biotransformation and cell death accompanied the cessation of TCG accumulation in cultures. The results suggest that TCG is the mediator of a gradual deterioration in the state of the culture which leads to a loss of both in vivo and in vitro toluene dioxygenase activity and a marked decrease in culture viability.
TL;DR: It is proposed that the region encodes xylene oxygenase activity capable of direct monohydroxylation of indole to 3-hydroxyindole (oxindole), which then spontaneously dimerizes to form indigo.
Abstract: The entire operon coding for the enzymes responsible for conversion of toluenes to benzoates has been cloned from TOL plasmid pWW53 and the position of the genes accurately located. The coding region was 7.4 kilobase pairs (kbp) long, and the gene order was operator-promoter region (OP1)-a small open reading frame-xylC (1.6 kbp)-xylA (2.9 kbp)-xylB (1.8 kbp). Within the coding region there was considerable homology with the isofunctional region of the archetypal TOL plasmid pWW0. A central region of 2.9 kbp complemented an xylA (for xylene oxygenase) mutant of Pseudomonas putida mt-2 and was also capable of conferring the ability to convert indole to indigo on strains of Escherichia coli and P. putida. This reaction has been reported previously only for dioxygenases involved in aromatic catabolism but not for monooxygenases. It is proposed that the region encodes xylene oxygenase activity capable of direct monohydroxylation of indole to 3-hydroxyindole (oxindole), which then spontaneously dimerizes to form indigo. Images
TL;DR: A two-stage continuous culture system to overcome the impact of the high-level expression of the xylE gene on the stability of pTG201 is described, where immobilized cells were grown in the repressed state in order to prevent loss of p TG201.
Abstract: SUMMARY: The effect of temperature on the stability of pTG201, a plasmid carrying the xylE gene (which encodes catechol 2,3-dioxygenase from Pseudomonas putida), and the production of catechol 2,3-dioxygenase in free and immobilized Escherichia coli during continuous culture have been studied at various temperatures. Immobilization of cells increased the stability of pTG201 considerably, even under conditions when expression of the xylE product was enhanced. Since xylE transcription was controlled by the γPR promoter and cI857 repressor, increasing derepression temperatures increased catechol 2,3-dioxygenase productivity and decreased pTG201 stability. A two-stage continuous culture system to overcome the impact of the high-level expression of the xylE gene on the stability of pTG201 is described. In the first stage, immobilized cells were grown in the repressed state in order to prevent loss of pTG201, whereas in the second stage, cultures were maintained in the derepressed state.