Showing papers on "Pseudomonas putida published in 1991"
TL;DR: In this article, a 25-kilobase (kb) DNA fragment was isolated from Pseudomonas oleovorans and used to select DNA fragments which encode poly(3-hydroxyalkanoates) (PHAs) and a PHA depolymerase.
278 citations
TL;DR: A helix-turn-helix motif is predicted in the most highly-conserved segment of each protein suggesting that they are members of a new family of helix/turn/helix DNA-binding proteins.
Abstract: A new family of bacterial regulatory proteins has been identified by sequence similarity. The family contains the repressor of the Bacillus subtilis gluconate operon (GntR), the regulators for histidine utilization in Pseudomonas putida (HutCPp) and Klebsiella aerogenes (HutCKa), the repressor (FadR) of fatty acid degradation in Escherichia coli, a regulator involved in the conjugal transfer of the broad host range plasmid pIJ101 (KorA), and three proteins of unidentified function in E. coli (GenA, P30 and PhnF). The proteins share amino acid sequence similarities in a 69-residue N-terminal region. A helix-turn-helix motif is predicted in the most highly-conserved segment of each protein suggesting that they are members of a new family of helix-turn-helix DNA-binding proteins.
228 citations
TL;DR: Pseudomonas fluorescens, Pseudomona putida, PseUDomonas syringae, and pseudomonas mendocina all had a functional analogue of ANR, indicating that similar anaerobic control mechanisms exist in these bacteria.
Abstract: Summary
Anaerobic growth of Pseudomonas aeruginosa on nitrate or arginine requires the anr gene, which codes for a positive control element (ANR) capable of functionally complementing an fnr mutation in Escherichia coli. The anr gene was sequenced; it showed 51% identity with the fnr gene at the amino acid sequence level. Four cysteine residues known to be essential in the FNR protein are conserved in ANR. The anr gene product (deduced Mr 27129) was visualized by the maxicell method and migrated like a 32kDa protein in gel electrophoresis under denaturing conditions. An anr mutant of P. aeruginosa constructed by gene replacement was defective in nitrate respiration, arginine deiminase activity, and hydrogen cyanide biosynthesis, underscoring the diverse metabolic functions of ANR during oxygen limitation. Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas syringae, and Pseudomonas mendocina all had a functional analogue of ANR, indicating that similar anaerobic control mechanisms exist in these bacteria.
191 citations
TL;DR: Measurements of the specific activities of chlorocatechol 1,2-dioxygenases encoded by tcbC, clcA, and tfdC suggested that a specialization among type II enzymes has taken place.
Abstract: Pseudomonas sp. strain P51 contains two gene clusters located on catabolic plasmid pP51 that encode the degradation of chlorinated benzenes. The nucleotide sequence of a 5,499-bp region containing the chlorocatechol-oxidative gene cluster tcbCDEF was determined. The sequence contained five large open reading frames, which were all colinear. The functionality of these open reading frames was studied with various Escherichia coli expression systems and by analysis of enzyme activities. The first gene, tcbC, encodes a 27.5-kDa protein with chlorocatechol 1,2-dioxygenase activity. The tcbC gene is followed by tcbD, which encodes cycloisomerase II (39.5 kDa); a large open reading frame (ORF3) with an unknown function; tcbE, which encodes hydrolase II (25.8 kDa); and tcbF, which encodes a putative trans-dienelactone isomerase (37.5 kDa). The tcbCDEF gene cluster showed strong DNA homology (between 57.6 and 72.1% identity) and an organization similar to that of other known plasmid-encoded operons for chlorocatechol metabolism, e.g., clcABD of Pseudomonas putida and tfdCDEF of Alcaligenes eutrophus JMP134. The identity between amino acid sequences of functionally related enzymes of the three operons varied between 50.6 and 75.7%, with the tcbCDEF and tfdCDEF pair being the least similar of the three. Measurements of the specific activities of chlorocatechol 1,2-dioxygenases encoded by tcbC, clcA, and tfdC suggested that a specialization among type II enzymes has taken place. TcbC preferentially converts 3,4-dichlorocatechol relative to other chlorinated catechols, whereas TfdC has a higher activity toward 3,5-dichlorocatechol. ClcA takes an intermediate position, with the highest activity level for 3-chlorocatechol and the second-highest level for 3,5-dichlorocatechol.
160 citations
TL;DR: A fragment from one of the cosmid clones was subcloned into pUC118, and subsequent deletion and gas chromatography-mass spectrometry analyses revealed that a fragment of ca.
Abstract: Pseudomonas paucimobilis UT26 is capable of growing on gamma-hexachlorocyclohexane (gamma-HCH). A genomic library of P. paucimobilis UT26 was constructed in Pseudomonas putida by using the broad-host-range cosmid vector pKS13. After 2,300 clones were screened by gas chromatography, 3 clones showing gamma-HCH degradation were detected. A 5-kb fragment from one of the cosmid clones was subcloned into pUC118, and subsequent deletion and gas chromatography-mass spectrometry analyses revealed that a fragment of ca. 500 bp was responsible for the conversion of gamma-HCH to 1,2,4-trichlorobenzene via gamma-pentachlorocyclohexene. Nucleotide sequence analysis revealed an open reading frame (linA) of 465 bp within the fragment. The nucleotide sequence of the linA gene and the deduced amino acid sequence showed no similarity to any known sequences. The product of the linA gene was 16.5 kDa according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Images
156 citations
TL;DR: Iron uptake from ferrated (59Fe) pseudobactin (PSB), a Pseudomonas putida siderophore, by various plant species was studied in nutrient solution culture under short term and long term conditions.
Abstract: Iron uptake from ferrated (59Fe) pseudobactin (PSB), a Pseudomonas putida siderophore, by various plant species was studied in nutrient solution culture under short term (10 h) and long term (3 weeks) conditions. In the short term experiments, 59Fe uptake rate from 59FePSB by dicots (peanuts, cotton and sunflower) was relatively low when compared with 59Fe uptake rate from 59FeEDDHA. Iron uptake rate from 59FePSB was pH and concentration dependent, as was the Fe uptake rate from 59FeEDDHA. The rate was about 10 times lower than that of Fe uptake from the synthetic chelate. Results were similar for long term experiments.
148 citations
TL;DR: Two gene clusters involved in the degradation of these compounds were identified on a catabolic plasmid, pP51, by using hybridization and characterized by cloning in Escherichia coli, Pseudomonas putida KT2442, and Alcaligenes eutrophus JMP222.
Abstract: Pseudomonas sp. strain P51 is able to use 1,2-dichlorobenzene, 1,4-dichlorobenzene, and 1,2,4-trichlorobenzene as sole carbon and energy sources. Two gene clusters involved in the degradation of these compounds were identified on a catabolic plasmid, pP51, with a size of 110 kb by using hybridization. They were further characterized by cloning in Escherichia coli, Pseudomonas putida KT2442, and Alcaligenes eutrophus JMP222. Expression studies in these organisms showed that the upper-pathway genes (tcbA and tcbB) code for the conversion of 1,2-dichlorobenzene and 1,2,4-trichlorobenzene to 3,4-dichlorocatechol and 3,4,6-trichlorocatechol, respectively, by means of a dioxygenase system and a dehydrogenase. The lower-pathway genes have the order tcbC-tcbD-tcbE and encode a catechol 1,2-dioxygenase II, a cycloisomerase II, and a hydrolase II, respectively. The combined action of these enzymes degrades 3,4-dichlorocatechol and 3,4,6-trichlorocatechol to a chloromaleylacetic acid. The release of one chlorine atom from 3,4-dichlorocatechol takes place during lactonization of 2,3-dichloromuconic acid.
144 citations
TL;DR: Analysis of one such pattern suggests that mutations creating different DNA slippage structures made a significant contribution to the evolutionary divergence of xylX, and this region is carried on the TOL pWW0 plasmid in Pseudomonas putida.
Abstract: The xylXYZ DNA region is carried on the TOL pWW0 plasmid in Pseudomonas putida and encodes a benzoate dioxygenase with broad substrate specificity. The DNA sequence of the region is presented and compared with benABC, the chromosomal region encoding the benzoate dioxygenase of Acinetobacter calcoaceticus. Corresponding genes from the two biological sources share common ancestry: comparison of aligned XylX-BenA, XylY-BenB, and XylZ-BenC amino acid sequences revealed respective identities of 58.3, 61.3, and 53%. The aligned genes have diverged to assume G+C contents that differ by 14.0 to 14.9%. Usage of the unusual arginine codons AGA and AGG appears to have been selected in the P. putida xylX gene as it diverged from the ancestor it shared with A. calcoaceticus benA. Homologous A. calcoaceticus and P. putida genes exhibit different patterns of DNA sequence repetition, and analysis of one such pattern suggests that mutations creating different DNA slippage structures made a significant contribution to the evolutionary divergence of xylX.
134 citations
TL;DR: Results indicate that the nitrobenzene ring is subject to initial attack by both mono- and dioxygenase enzymes.
Abstract: Nonpolar nitroaromatic compounds have been considered resistant to attack by oxygenases because of the electron withdrawing properties of the nitro group. We have investigated the ability of seven bacterial strains containing toluene degradative pathways to oxidize nitrobenzene. Cultures were induced with toluene vapor prior to incubation with nitrobenzene, and products were identified by high-performance liquid chromatography and gas chromatography-mass spectrometry. Pseudomonas cepacia G4 and a strain of Pseudomonas harboring the TOL plasmid (pTN2) did not transform nitrobenzene. Cells of Pseudomonas putida F1 and Pseudomonas sp. strain JS150 converted nitrobenzene to 3-nitrocatechol. Transformation of nitrobenzene in the presence of 18O2 indicated that the reaction in JS150 involved the incorporation of both atoms of oxygen in the 3-nitrocatechol, which suggests a dioxygenase mechanism. P. putida 39/D, a mutant strain of P. putida F1, converted nitrobenzene to a compound tentatively identified as cis-1,2-dihydroxy-3-nitrocyclohexa-3,5-diene. This compound was rapidly converted to 3-nitrocatechol by cells of strain JS150. Cultures of Pseudomonas mendocina KR-1 converted nitrobenzene to a mixture of 3- and 4-nitrophenol (10 and 63%, respectively). Pseudomonas pickettii PKO1 converted nitrobenzene to 3- and 4-nitrocatechol via 3- and 4-nitrophenol. The nitrocatechols were slowly degraded to unidentified metabolites. Nitrobenzene did not serve as an inducer for the enzymes that catalyzed its oxidation. These results indicate that the nitrobenzene ring is subject to initial attack by both mono- and dioxygenase enzymes.
122 citations
TL;DR: The amino acid sequence between positions 8 and 37 of salicylate hydroxylase shows homology with known ADP binding sites of other FAD-containing oxidoreductases, thus confirming its biochemical function.
Abstract: Gene nahG of naphthalene/salicylate catabolic plasmid NAH7 encodes a protein of molecular weight 45,000, salicylate hydroxylase. This enzyme catalyzes the formation of catechol from salicylate, a key intermediate in naphthalene catabolism. DNA sequence analysis of the 3.1-kilobase HindIII fragment containing the nahG locus reveals an open reading frame (ORF) of 1305 base pairs that corresponds to a protein of 434 amino acid residues. The predicted amino acid sequence of salicylate hydroxylase is in agreement with the molecular weight, NH2-terminal amino acid sequence, and total amino acid composition of the purified salicylate hydroxylase [You, I.-S., Murray, R. I., Jollie, D., & Gunsalus, I. C. (1990) Biochem. Biophys. Res. Commun. 169, 1049-1054]. The amino acid sequence between positions 8 and 37 of salicylate hydroxylase shows homology with known ADP binding sites of other FAD-containing oxidoreductases, thus confirming its biochemical function. The sequence of the Pseudomonas putida salicylate hydroxylase was compared with those of other similar flavoproteins. A small DNA segment (831 base pairs) disrupts the continuity of the known gene order nahG and nahH, the latter encoding catechol 2,3-dioxygenase. The complete nucleotide sequence of the intergenic region spanning genes nahG and nahH has been determined and its biological role proposed.
121 citations
TL;DR: The recombinant pWW0-EB62 plasmid and its catabolic functions were stable for periods of up to 1 month in bacteria introduced in unamended soils and only conferred selective advantage to the host bacteria without theplasmid or with the natural pWW 0 plasmids when the soils were amended with low amounts of p-ethylbenzoate.
Abstract: Pseudomonas putida EEZ15(pWW0-EB62) is a phosphinothricin (PPT)-resistant strain with a recombinant TOL plasmid which allows the strain to grow on p-ethylbenzoate. The survival of this strain in sterile agricultural soils depends on the physicochemical properties of the soil. The recombinant pWW0-EB62 plasmid and its catabolic functions were stable for periods of up to 1 month in bacteria introduced in unamended soils and only conferred selective advantage to the host bacteria without the plasmid or with the natural pWW0 plasmid when the soils were amended with low amounts of p-ethylbenzoate. The addition to soils of aromatics that are cometabolized by P. putida EEZ15(pWW0-EB62) had a detrimental effect on the survival of the bacteria, whereas low amounts of aromatics that are not metabolized by this bacterium had no effect on their survival. Survival of P. putida EEZ15(pWW0-EB62) was better at 4 and 25 degrees C than at 37 degrees C. The host bacterium carrying the recombinant pWW0-EB62 plasmid was established in unsterile soils.
TL;DR: Compared to mesophilic pseudomonads previously studied, the psychrotrophic strain grows on and degrades phenol at rates that are ca.
Abstract: Cell growth and phenol degradation kinetics were studied at 10°C for a psychrotrophic bacterium, Pseudomonas putida Q5. The batch studies were conducted for initial phenol concentrations, So, ranging from 14 to 1000 mg/1. The experimental data for 14<=So<=200 mg/1 were fitted by non-linear regression to the integrated Haldane substrate inhibition growth rate model. The values of the kinetic parameters were found to be: μm=0.119 h−1, KS=5.27 mg/1 and KI=377 mg/1. The yield factor of dry biomass from substrate consumed was Y=0.55. Compared to mesophilic pseudomonads previously studied, the psychrotrophic strain grows on and degrades phenol at rates that are ca. 65–80% lower. However, use of the psychrotrophic microorganism may still be economically advantageous for waste-water treatment processes installed in cold climatic regions, and in cases where influent waste-water temperatures exhibit seasonal variation in the range 10–30°C.
TL;DR: It is concluded that PupA is a specific receptor for ferric pseudobactin 358, and that strain WCS358 produces at least one other receptor for other pseudobactsins.
Abstract: Summary
The initial step in the uptake of iron via ferric pseudobactin by the plant-growth-promoting Pseudomonas putida strain WCS358 is binding to a specific outer-membrane protein. The nucleotide sequence of the pupA structural gene, which codes for a ferric pseudobactin receptor, was determined. It contains a single open reading frame which potentially encodes a polypeptide of 819 amino acids, including a putative N-terminal signal sequence of 47 amino acids. Significant homology, concentrated in four boxes, was found with the TonB-dependent receptor proteins of Escherichia coli. The pupA mutant MH100 showed a residual efficiency of 30% in the uptake of 55HFe3+ complexed to pseudobactin 358, whereas the iron uptake of four other pseudobactins was not reduced at all. Cells of strain WCS374 supplemented with the pupA gene of strain WCS358 could transport ferric pseudobactin 358 but showed no affinity for three other pseudobactins. It is concluded that PupA is a specific receptor for ferric pseudobactin 358, and that strain WCS358 produces at least one other receptor for other pseudobactins.
TL;DR: The presence of the catabolic genes tcbA and tcbB on Tn5280 suggests a mechanism by which gene clusters can be mobilized as gene cassettes and joined with others to form novel catabolic pathways.
Abstract: Analysis of one of the regions of catabolic plasmid pP51 which encode chlorobenzene metabolism of Pseudomonas sp. strain P51 revealed that the tcbA and tcbB genes for chlorobenzene dioxygenase and dehydrogenase are located on a transposable element, Tn5280. Tn5280 showed the features of a composite bacterial transposon with iso-insertion elements (IS1066 and IS1067) at each end of the transposon oriented in an inverted position. When a 12-kb HindIII fragment of pP51 containing Tn5280 was cloned in the suicide donor plasmid pSUP202, marked with a kanamycin resistance gene, and introduced into Pseudomonas putida donor plasmid pSUP202, marked with a kanamycin resistance gene, and introduced into Pseudomonas putida KT2442, Tn5280 was found to transpose into the genome at random and in single copy. The insertion elements IS1066 and IS1067 differed in a single base apir located in the inner inverted repeat and were found to be highly homologous to a class of repetitive elements of Bradyrhizobium japonicum and distantly related to IS630 of Shigella sonnei. The presence of the catabolic genes tcbA and tcbB on Tn5280 suggests a mechanism by which gene clusters can be mobilized as gene cassettes and joined with others to form novel catabolic pathways. Images
TL;DR: Intraspecies transfer of TOL in soils was affected by the type of soil used, the initial inoculum size, and the presence of chemicals that could affect the survival of the donor or recipient bacteria.
Abstract: Recombinant TOL plasmid pWWO-EB62 allows Pseudomonas putida to grow on p-ethylbenzoate. This plasmid can be transferred to other microorganisms, and its catabolic functions for the metabolism of alkylbenzoates are expressed in a limited number of gram-negative bacteria, including members of pseudomonad rRNA group I and Escherichia coli. Transfer of the recombinant plasmid to Erwinia chrysanthemi was observed, but transconjugants failed to grow on alkylbenzoates because they lost catabolic functions. Pseudomonads belonging to rRNA groups II, III, and IV, Acinetobacter calcoaceticus, and Alcaligenes sp. could not act as recipients for TOL, either because the plasmid was not transferred or because it was not stably maintained. The frequency of transfer of pWWO-EB62 from P. putida as a donor to pseudomonads belonging to rRNA group I was on the order of 1 to 10(-2) transconjugant per recipient, while the frequency of intergeneric transfer ranged from 10(-3) to 10(-7) transconjugant per recipient. The profile of potential hosts was conserved when the donor bacterium was Escherichia coli or Erwinia chrysanthemi instead of P. putida. No intergeneric gene transfer of the recombinant TOL plasmid was observed in soils; however, intraspecies transfer did take place. Intraspecies transfer of TOL in soils was affected by the type of soil used, the initial inoculum size, and the presence of chemicals that could affect the survival of the donor or recipient bacteria. Images
TL;DR: Although pheA and pheB are cotranscribed using the promoter sequences derived from Tn4652 and the level of expression of C120 activities from pEST1412 was equal both in Escherichia coli and in Pseudomonas putida, thelevel of PMO activity measured in the cell-free extracts of E. coli was lower than that in P. putida.
TL;DR: The results reported in this paper illustrate the importance of choosing strains which are well adapted to environmental conditions if the use of microbial inoculants for the breakdown of target pollutants is to be successful.
Abstract: The survival of selected naturally occurring and genetically engineered bacteria in a fully functional laboratory-scale activated-sludge unit (ASU) was investigated. The effect of the presence of 3-chlorobenzoate (3CB) on the survival of Pseudomonas putida UWC1, with or without a chimeric plasmid, pD10, which encodes 3CB catabolism, was determined. P. putida UWC1(pD10) did not enhance 3CB breakdown in the ASU, even following inoculation at a high concentration (3 x 10(8) CFU/ml). The emergence of a natural, 3CB-degrading population appeared to have a detrimental effect on the survival of strain UWC1 in the ASU. The fate of two 3CB-utilizing bacteria, derived from activated-sludge microflora, was studied in experiments in which these strains were inoculated into the ASU. Both strains, AS2, an unmanipulated natural isolate which flocculated readily in liquid media, and P. putida ASR2.8, a transconjugant containing the recombinant plasmid pD10, survived for long periods in the ASU and enhanced 3CB breakdown at 15 degrees C. The results reported in this paper illustrate the importance of choosing strains which are well adapted to environmental conditions if the use of microbial inoculants for the breakdown of target pollutants is to be successful.
TL;DR: The gene (todF) encoding 2-hydroxy-6-oxohepta-2,4-dienoate hydrolase in Pseudomonas putida F1 was shown to be located upstream of the todC1C2BADE genes, which encode the enzymes responsible for the initial reactions in toluene degradation.
TL;DR: A Pseudomonas putida strain able to grow in the presence of more than 50% toluene was isolated from soil and was tolerant of other toxic solvents, including aliphatic hydrocarbons, alicyclic hydro Carbons, aromatic hydrocarsbons, alcohols, and ethers.
Abstract: A Pseudomonas putida strain able to grow in the presence of more than 50% toluene was isolated from soil. The strain was tolerant of other toxic solvents, including aliphatic hydrocarbons, alicyclic hydrocarbons, aromatic hydrocarbons, alcohols, and ethers. The stability of the solvent tolerance of strain IH-2000 was stimulated by addition of Mg2+ and Ca2+ to the medium containing toluene.
TL;DR: Amino acid sequence comparisons indicated that TcbR is a member of the LysR family of transcriptional activator proteins and shares a high degree of homology with other activators involved in regulating the metabolism of aromatic compounds.
Abstract: Plasmid pP51 of Pseudomonas sp. strain P51 contains two gene clusters encoding the degradation of chlorinated benzenes, tcbAB and tcbCDEF. A regulatory gene, tcbR, was located upstream and divergently transcribed from the chlorocatechol oxidative gene cluster tcbCDEF. The tcbR gene was characterized by DNA sequencing and expression studies with Escherichia coli and pET8c and appeared to encode a 32-kDa protein. The activity of the tcbR gene product was analyzed in Pseudomonas putida KT2442, in which it appeared to function as a positive regulator of tcbC expression. Protein extracts of both E. coli overproducing TcbR and Pseudomonas sp. strain P51 showed specific DNA binding to the 150-bp region that is located between the tcbR and tcbC genes. Primer extension mapping demonstrated that the transcription start sites of tcbR and tcbC are located in this region and that the divergent promoter sequences of both genes overlap. Amino acid sequence comparisons indicated that TcbR is a member of the LysR family of transcriptional activator proteins and shares a high degree of homology with other activator proteins involved in regulating the metabolism of aromatic compounds.
TL;DR: In vitro and in situ assays demonstrated that 2,4-DCP was toxic to fungal propagules at concentrations below those detected in the soil and did not appear to depress the numbers of total heterotrophic, sporeforming, or chitin-utilizing bacteria.
Abstract: A genetically engineered microorganism, Pseudomonas putida PPO301(pRO103), and the plasmidless parent strain, PPO301, were added at approximately 107 CFU/g of soil amended with 500 ppm of 2,4-dichlorophenoxyacetate (2,4-D) (500 μg/g). The degradation of 2,4-D and the accumulation of a single metabolite, identified by gas chromatography-mass spectrophotometry as 2,4-dichlorophenol (2,4-DCP), occurred only in soil inoculated with PPO301(pRO103), wherein 2,4-DCP accumulated to >70 ppm for 5 weeks and the concentration of 2,4-D was reduced to 400-fold decline in the numbers of fungal propagules and a marked reduction in the rate of CO2 evolution, whereas 2,4-D did not depress either fungal propagules or respiration of the soil microbiota. 2,4-DCP did not appear to depress the numbers of total heterotrophic, sporeforming, or chitin-utilizing bacteria. In vitro and in situ assays conducted with 2,4-DCP and fungal isolates from the soil demonstrated that 2,4-DCP was toxic to fungal propagules at concentrations below those detected in the soil.
TL;DR: Pseudomona cepacia R55 and R85 and Pseudomonas putida R104, antagonistic towards plant pathogenic fungi in vitro, were assessed as seed inoculants for winter wheat grown in a growth chamber in soil infested with Fusarium solani or Rhizoctonia solani isolate AG-1, AG 2-1.
Abstract: Pseudomonas cepacia R55 and R85 and Pseudomonas putida R104, antagonistic towards plant pathogenic fungi in vitro, were assessed as seed inoculants for winter wheat (cv. Norstar) grown in a growth ...
TL;DR: Hyphal growth but not sporangial germination of Pythium ultimum was inhibited by the synthetic iron chelating compound ethylenediamine on water agar and by strain N1R of Pseudomonas putida on King's medium B.
Abstract: Hyphal growth but not sporangial germination of Pythium ultimum was inhibited by the synthetic iron chelating compound ethylenediamine (di-o-hydroxyphenylacetic acid) (EDDHA) on water agar and by strain N1R of Pseudomonas putida on King's medium B. Eight prototrophic derivatives of N1R (deficient in fluorescent siderophore [pyoverdine] biosynthesis [Pvd - ]) were obtained by Tn5 mutagenesis (...)
TL;DR: 3,5-dichlorobenzoate completely inhibited growth of P111 on all ortho-substituted benzoates that were tested, and it is apparent that 1-carboxy-1,2-dihydroxy-3, 5- dichlorocyclohexadiene is not further metabolized by these cells.
Abstract: Pseudomonas putida P111 was isolated by enrichment culture on 2,5-dichlorobenzoate and was also able to grow on 2-chloro-, 3-chloro-, 4-chloro-, 2,3-dichloro-, 2,4-dichloro-, and 2,3,5-trichlorobenzoates. However, 3,5-dichlorobenzoate completely inhibited growth of P111 on all ortho-substituted benzoates that were tested. When 3,5-dichlorobenzoate was added as a cosubstrate with either 3- or 4-chlorobenzoate, cell yields and chloride release were greater than those observed from growth on either monochlorobenzoate alone. Moreover, resting cells of P111 grown on 4-chlorobenzoate released chloride from 3,5-dichlorobenzoate and produced no identifiable intermediate. In contrast, resting cells grown on 2,5-dichlorobenzoate metabolized 3,5-dichlorobenzoate without release of chloride and accumulated a degradation product, which was identified as 1-carboxy-1,2-dihydroxy-3,5-dichlorocyclohexadiene on the basis of gas chromatography-mass spectrometry confirmation of its two acid-hydrolyzed products, 3,5- and 2,4-dichlorophenol. Since 3,5-dichlorocatechol was rapidly metabolized by cells grown on 2,5-dichlorobenzoate, it is apparent that 1-carboxy-1,2-dihydroxy-3,5-dichlorocyclohexadiene is not further metabolized by these cells. Moreover, induction of a functional dihyrodiol dehydrogenase would not be required for growth of P111 on other ortho-chlorobenzoates since the corresponding chlorodihydrodiols produced from a 1,2-dioxygenase attack would spontaneously decompose to the corresponding catechols. In contrast, growth on 3-chloro-, 4-chloro-, or 3,5-dichlorobenzoate requires a functional dihydrodiol dehydrogenase, yet only the two monochlorobenzoates appear to induce for it.
TL;DR: It will be difficult to predict accurately the survival of released populations in the natural environment, as a large variability was found depending on which water sample was used.
Abstract: IncQ marker plasmids were previously constructed to enable the analysis of the survival of populations of Pseudomonas putida released into lake water (C. Winstanley, J. A. W. Morgan, R. W. Pickup, J. G. Jones, and J. R. Saunders, Appl. Environ. Microbiol. 55:771-777, 1989). We constructed equivalent IncP plasmids, pLV1016 and pLV1017, to provide conjugative alternative systems. Detection of the xylE gene carried by marker plasmids was found to be a valid indicator to use for studying the survival of released populations by culturing on nonselective media. These plasmids were used to study the survival of populations of Pseudomonas putida in both sterile and untreated lake water. The effects of inoculum size, the metabolic burden imposed on the cell by the unregulated expression of xylE, and an auxotrophic mutation carried by the host strain were studied. We also assessed the reproducibility and hence the predictability of the survival of released populations. Model systems with a single lake water sample and model systems with three different lake water samples, taken from the same site in consecutive months, were used to analyze variability between replicates and to assess differences caused by host strain or water sample. A large variability was found depending on which water sample was used. These findings imply that it will be difficult to predict accurately the survival of released populations in the natural environment.
TL;DR: Since the beads and bead-cell complexes were recovered in a magnetic field, target bacteria were separated from other lake water organisms and from particulate material which was not attracted towards the magnet and were thereby enriched and may now provide a useful system for recovering recombinant bacteria selectively from environmental samples.
Abstract: Monoclonal antibodies to Pseudomonas putida Paw340 cells were produced. In an enzyme-linked immunosorbent assay (ELISA) against whole bacterial cells, a hybridoma cell line termed MLV1 produced a monoclonal antibody that reacted with P. putida Paw340 but showed no cross-reaction with 100 medical isolates and 150 aquatic isolates. By ELISA, immunogold electron microscopy, and Western blot (immunoblot) analysis, MLV1 antibody was found to react with purified bacterial flagella. The surfaces of magnetic polystyrene beads were coated with MLV1 antibody. By mixing MLV1 antibody-coated beads with lake water samples containing the target P. putida host, bead-cell complexes which could be recovered by attraction towards a magnet were formed. Prevention of nonspecific attachment of cells to the beads required the incorporation of detergents in the isolation protocol. These detergents affected colony-forming ability; however, the cells remained intact for direct detection. When reisolated by standard cultural methods, approximately 20% of the initial target population was recovered. Since the beads and bead-cell complexes were recovered in a magnetic field, target bacteria were separated from other lake water organisms and from particulate material which was not attracted towards the magnet and were thereby enriched. This method may now provide a useful system for recovering recombinant bacteria selectively from environmental samples.
TL;DR: The effect of a basidiome stimulatory Pseudomonas putida isolate on hyphal growth of the cultivated mushroom, Agaricus bisporus, and the effect of P. putida on dry weight of fungal material was investigated.
TL;DR: There are conserved residues in all the four protein products of these genes, including pheB, which encodes a protein product with a deduced Mr of 33,362 and other C12O-encoding genes have been shown.
TL;DR: Investigations into the nature of the attractants contained in the mycelial exudate indicated that they are predominantly small (Mr less than 2000) thermostable compounds.
Abstract: SUMMARY: The chemotactic response of wild-type Pseudomonas putida and P tolaasii, and a phenotypic variant of each species, to Agaricus bisporus mycelial exudate was examined Both P putida, the bacterium responsible for initiating basidiome development of A bisporus, and P tolaasii, the causal organism of bacterial blotch disease of the mushroom, displayed a positive chemotactic response to Casamino acids and to A bisporus mycelial exudate The response was both dose- and time-dependent and marked differences were observed between the response time of the wild-type strains and their phenotypic variants Phenotypic variants responded rapidly to both attractants and reached a maximum response after 10-20 min, whereas the wild-types took 45-60 min The differences are partly explained by the more rapid swimming speed of the phenotypic variants Both variants responded maximally to similar concentrations of Casamino acids and mycelial exudates Investigations into the nature of the attractants contained in the mycelial exudate indicated that they are predominantly small (M
r > 2000) thermostable compounds Sugars present in the exudate did not elicit a chemotactic response in any isolate, but a mixture of 14 amino acids detected in the exudate accounted for between 50 and 75% of the chemotactic response of the fungal exudate
TL;DR: A 43,000 molecular-weight, glucose-inducible, organic acid-repressible protein (OprB) was identified in the outer membrane of Pseudomonas putida and appeared to be distinct from that of the maltodextrin-specific porin LamB from Escherichia coli.
Abstract: A 43,000 molecular-weight, glucose-inducible, organic acid-repressible protein (OprB) was identified in the outer membrane of Pseudomonas putida. OprB was surface expressed in whole cells, had a high beta-sheet content, and was heat modifiable, as demonstrated by 125I-labeling, circular dichroism spectroscopy, and mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. OprB was extracted from outer membrane preparations by using 2% Lubrol PX with 10 mM EDTA and purified by DEAE-Sephacel ion exchange chromatography following ammonium sulfate precipitation. Reconstitution experiments with black lipid membranes showed that OprB formed small, cation-selective pores which bound glucose (KS = 110 mM) and other carbohydrates. However, the binding site of OprB appeared to be distinct from that of the maltodextrin-specific porin LamB from Escherichia coli.