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At least 217 records · Page 12Linked to original sources

PubMed1992

Luminometric measurement of population activity of genetically modified Pseudomonas fluorescens in the soil.

Genetically modified cells of Pseudomonas fluorescens, chromosomally marked with genes for bioluminescence, were inoculated into sterile soil microcosms. During incubation for 90 days, viable cell concentration did not change significantly but light output, measured by luminometry, decreased, indicating reduced metabolic activity due to lack of substrates. Amendment with nutrients resulted in parallel increases in both luminescence and dehydrogenase activity. Luminometry therefore enables rapid monitoring of the activity of populations of luminescence-marked microbial inocula in the soil, with greater sensitivity and selectivity than traditional techniques.

Bacteriological Techniques↗

PubMed1976

Methionine degradation by Pseudomonas fluorescens UK1 and its methionine-utilizing mutant.

Pseudomonas fluorescens UK1, and a mutant derived from it that can use methionine as its sole source of carbon and nitrogen, were used to study methionine degradation. Radioactive 2-oxomethionine, 2-oxobutyric acid and carbon dioxide comprised 95% of [I-14C]methionine consumed by the mutant. Demethiolating activity was detected in both strains when they were grown with methionine. The ability to grow on methionine depended on the decarboxylation of 2-oxobutyric acid derived from it. This decarboxylating activity was lost if the growth media contained an additional carbon source. No evidence was obtained for the operation of the cystathionine pathway.

Butyrates↗

PubMed1987

Peptidases from two strains of Pseudomonas fluorescens: partial purification, properties and action on milk.

Pseudomonas fluorescens strains 240 and 32A expressed cell-associated peptidase activity which was shown by subcellular fractionation to be primarily intracellular. Two peptidases were partly purified from strain 32A. One specifically hydrolysed N-alpha-benzoyl-DL-arginine-4-nitroanilide and was termed endopeptidase and the other hydrolysed L-lysine- and L-leucine-4-nitroanilide and was termed aminopeptidase. The endopeptidase had very low activity on bovine serum albumin compared with that of trypsin and probably was not a proteinase. The endopeptidase had a mol. wt of 33,000 and a pH optimum of 8.0. The enzyme was stimulated by Ca2+ and Mg2+ and inhibited by Co2+, Mn2+, Hg2+, Zn2+ and leupeptin. Soya bean trypsin inhibitor and phenylmethane sulphonyl fluoride (PMSF) had no effect on its activity. The aminopeptidase had a mol. wt of 44,000 and a pH optimum of 8.0. It was inhibited by all the metal ions mentioned above and by PMSF. Little proteolysis was found when ultra high temperature (UHT) sterilized milk was treated with cell-free extract from strain 32A. It was concluded that the cell-associated peptidases from Pseudomonas strains normally present in raw milk may not contribute significantly to the deterioration of UHT sterilized milk.

Aminopeptidases↗

PubMed1978

Transport of alpha-aminoisobutyrate by cells and membrane vesicles of Pseudomonas fluorescens.

The transport of alpha-aminoisobutyrate into Pseudomonas fluorescens NCIB 8865 and membrane vesicles prepared from this organism has been studied. Uptake by cells was mediated by two active transport systems with different apparent Km values, while transport into membrane vesicles was mediated by a single component. The effect of inhibitors on the energy-coupling mechanism for alpha-aminoisobutyrate transport in these systems suggests that a membrane potential may play a significant role in supporting alpha-aminoisobutyrate transport. The magnitude of the membrane potential in the vesicle system, and the sensitivity of its generation to inhibitors, has been measured using 137Cs in the presence of valinomycin. Direct attempts to demonstrate a protonsymport mechanism for alpha-aminoisobutyrate transport were negative.

Aminoisobutyric Acids↗

PubMed1993

Microbial transformations of ferulic acid by Saccharomyces cerevisiae and Pseudomonas fluorescens.

Saccharomyces cerevisiae (dry baker's yeast) and Pseudomonas fluorescens were used to convert trans-ferulic acid into 4-hydroxy-3-methoxystyrene in 96 and 89% yields, respectively. The metabolites were isolated by solid-phase extraction and analyzed by thin-layer chromatography and high-performance liquid chromatography. The identities of the metabolites were determined by 1H- and 13C-nuclear magnetic resonance spectroscopy and by mass spectrometry. The mechanism of the decarboxylation of ferulic acid was investigated by measuring the degree and position of deuterium incorporated into the styrene derivative from D2O by mass spectrometry and by both proton and deuterium nuclear magnetic resonance spectroscopies. Resting cells of baker's yeast reduced ferulic acid to 4-hydroxy-3-methoxyphenylpropionic acid in 54% yield when incubations were under an argon atmosphere.

Biotransformation↗

PubMedDate not supplied

[Comparative characteristics of lipopolysaccharides of various Pseudomonas fluorescens strains (Biovar I)].

From the biomass of five Pseudomonas fluorescens biovar I strains, including the P. fluorescens type strain IMV 4125 (ATCC 13525), lipopolysaccharides (LPS) were isolated (by extraction with a phenol-water mixture followed by repeated ultracentrifugation), as well as individual structural components of the LPS macromolecule: lipid A, the core oligosaccharide, and O-specific polysaccharide (O-PS). 3-Hydroxydecanoic, 2-hydroxydodecanoic, 3-hydroxydodecanoic, dodecanoic, hexadecanoic, octadecanoic, hexadecenoic, and octadecenoic fatty acids were present in lipid A of the LPS of all the strains studied. Glucosamine, ethanolamine, and phosphoethanolamine were revealed in the lipid A hydrophilic part of all of KDO, a trace amount of heptoses, ethanolamine, phosphoethanolamine, alanine, and phosphorus were identified as the main core components. Interstrain differences in the core oligosaccharide composition were revealed. Structural analysis showed that the O-PS of the type strain, as distinct from that of other strains, is heterogeneous and contains two types of repetitive units, including (1) three L-rhamnose residues (L-Rha), one 3-acetamide-3,6-dideoxy-D-galactose residue (D-Fuc3NAc) as a branching substitute of the L-rhamnan chain and (2) three L-Rha residues and two branching D-Fuc3NAc residues. The type strain is also serologically distinct from other biovar I strains due to the LPS O-chain structure, which is similar to those of the strains of the species Pseudomonas syringae, including the type strain. The data of structural analysis agree well with the results of immunochemical studies of LPS.

Lipopolysaccharides↗

PubMed1987

Influence of EDTA and metal ions on a metalloproteinase from Pseudomonas fluorescens biotype I.

The inactivation of a metalloproteinase from Pseudomonas fluorescens Biotype I with EDTA was investigated at 22 degrees C and 37 degrees C. At 22 degrees C proteolytic activity decreases linearly with time and an inactive apoenzyme is obtained by dialysis. Proteolytic activity can be restored with several metal-ions, Ca2+, Zn2+, Mg2+, Sr2+ and co2+ give the best results. Activity and substrate specificity are influenced by the metal-ions. Reactivation depends on the concentration of the metal-ions, optimum concentration is 1 mM for Ca2+ and 50 microM for Zn2+. The isoelectric point of the apoenzyme is around 8.0, this is about 0.3 pH-units lower than the isoelectric point of the native proteinase. At 37 degrees C inactivation follows first order kinetics and is irreversible because of autolysis as shown by a gel filtration-experiment.

Apoenzymes↗

PubMed2000

Elevated zinc induces siderophore biosynthesis genes and a zntA-like gene in Pseudomonas fluorescens.

Zinc-regulated genes were analyzed in Pseudomonas fluorescens employing mutagenesis with a reporter gene transposon. Six mutants responded with increased gene expression to elevated concentrations of zinc. Genetic and biochemical analysis revealed that in four of the six mutants the transposon had inserted into genes essential for the biosynthesis of the siderophore pyoverdine. The growth of one of the mutants was severely impaired in the presence of elevated concentrations of cadmium and zinc ions. In this mutant, the transposon had inserted in a gene with high similarity to P-type ATPases involved in zinc and cadmium ion transport. Four mutants reacted with reduced gene expression to elevated concentrations of zinc. One of these mutants was sensitive to zinc, cadmium and copper ions. The genetic region targeted in this mutant did not show similarity to any known gene.

Adenosine Triphosphatases↗

PubMed2006

Modeling high-intensity pulsed electric field inactivation of a lipase from Pseudomonas fluorescens.

The inactivation kinetics of a lipase from Pseudomonas fluorescens (EC 3.1.1.3.) were studied in a simulated skim milk ultrafiltrate treated with high-intensity pulsed electric fields. Samples were subjected to electric field intensities ranging from 16.4 to 27.4 kV/cm for up to 314.5 micros, thus achieving a maximum inactivation of 62.1%. The suitability of describing experimental data using mechanistic first-order kinetics and an empirical model based on the Weibull distribution function is discussed. In addition, different mathematical expressions relating the residual activity values to field strength and treatment time are supplied. A first-order fractional conversion model predicted residual activity with good accuracy (A(f) = 1.018). A mechanistic insight of the model kinetics was that experimental values were the consequence of different structural organizations of the enzyme, with uneven resistance to the pulsed electric field treatments. The Weibull model was also useful in predicting the energy density necessary to achieve lipase inactivation.

Animals↗

PubMedDate not supplied

[Substrate specificity, inhibitors and kinetics of deamidase AG (asparaginase-glutaminase) from Pseudomonas fluorescens AG].

Deamidase AG (asparaginase-glutaminase) from Pseudomonas fluorescens AG was shown to hydrolyze 1-glutamine and 1-asparagine highly effectively. Besides, the enzyme exhibited the rather high rate of deamidation of D-asparagine and D-glutamine (70% and 100%, respectively), Nalpha-butyl asparagine (63%) and among peptides -- of glycyl-L-asparagine (40%). L-glutamic acid gamma-methyl ester was hydrolyzed only slightly (5%). Effect of several substrate analogues on the deamidase AG activity was studied as well. Albiciine (alpha-amino-beta-ureide propionic acid) proved to be the strongest inhibitor (100%). Beta-Methyl aspartic acid, S-carbamoyl cysteine, alpha-ketoglutaric acid showed the slight inhibitory effect (20%). Amount of active centres per enzyme molecule was estimated by means of 14C-albiciine. Deamidase AG had apparently only one active centre. In estimation of relationship between the rate of reaction and substrate (L-asparagine) concentration, the reaction was found to follow Michaelis-Menten kinetics, K(m) = 4.5 with 10-4 M.

Asparaginase↗

PubMed1996

Aluminum detoxication mechanism in Pseudomonas fluorescens is dependent on iron.

The soil microbe Pseudomonas fluorescens has been shown to detoxify aluminum by the elaboration of a soluble metabolite where the trivalent metal is sequestered [Appanna and St. Pierre, FEMS Microbiol. Lett. 24 (1994) 327-332]. The inclusion of 5 mM iron in the growth medium elicited an entirely disparate detoxification strategy. In this instance, the two trivalent metals were immobilized in a gelatinous lipid-rich residue. Dialysis and ultracentrifugation studies indicated that the test metals were being transformed from early stages of growth and were associated with phosphatidylethanolamine. However, at 45 h of cellular multiplication, most of the metals were deposited as an insoluble residue. X-ray fluorescence analyses identified the constituents of this mineral essentially as aluminum, iron and phosphorus. Scanning electron microscopy and energy dispersive X-ray microanalysis of the dialysate, isolated at 35 h of microbial growth, revealed thread-like structures associated with nodule-like bodies that were rich in the two test metals. Transmission electron microscopic studies aided in the visualization of iron and aluminum inclusions within the bacterial cells.

Aluminum↗

PubMed1986

Multiple antibiotics produced by Pseudomonas fluorescens HV37a and their differential regulation by glucose.

Pseudomonas fluorescens HV37a inhibited growth of the fungus Pythium ultimum on potato dextrose agar (PDA). An antibiotic activity produced under these conditions was fractionated and partially characterized. Extracts prepared from the PDA on which HV37a was grown revealed a single peak of antibiotic activity on thin-layer chromatograms. Similar extracts were prepared from mutants of HV37a. Their analysis indicated that the antibiotic observed in thin-layer chromatograms was responsible for fungal inhibition observed on PDA. The production of the PDA antibiotic required the presence of glucose, whereas two other antibiotic activities were produced only on potato agar without added glucose. Two mutants (denoted AfuIa and AfuIb) previously characterized as deficient in fungal inhibition on PDA showed altered regulation of the production of all three antibiotics in response to glucose. These mutants were also deficient in glucose dehydrogenase. Mutants isolated as deficient in glucose dehydrogenase were also deficient in fungal inhibition and were grouped into two classes on the basis of complementation analysis with an AfuI cosmid. Glucose regulation of antibiotic biosynthesis therefore involves at least two components and requires glucose dehydrogenase.

Anti-Bacterial Agents↗

PubMed2004

Colonization and persistence of a plant growth-promoting bacterium Pseudomonas fluorescens strain CS85, on roots of cotton seedlings.

Pseudomonas fluorescens CS85, which was previously isolated from the rhizosphere of cotton seedlings, acts as both a plant growth-promoting bacterium and a biocontrol agent against cotton pathogens, including Rhizoctonia solani, Colletotrichum gossypii, Fusarium oxysporum f sp. vasinfectum, and Verticillium dahliae. Strain CS85 was labeled separately with luxAB and gusA. The labeled strains were stably maintained and had high levels of expression of the marker genes, luxAB and gusA, after successive transfers on nonselective medium, long-term preservation, and after recovery from soil. The labeled strains displayed similar biocontrol characteristics (e.g., antibiosis, effects of growth-promotion and disease-control) to the original strain. The labeled strains colonized all surfaces of the young plant root zones, such as roots hairs and lateral roots, although the distribution of the labeled strains on the root surfaces was not uniform. Moreover, the population densities of the labeled strains on the root surface were stably maintained at high levels during the first 2 weeks of plant growth in the native soil, so that about 10(7)-10(8) CFU/g root were detected, then decreased gradually. Nevertheless, approximately 10(6) CFU/g root of the labeled strains were observed on the root surfaces 35 d after planting.

Antibiosis↗

PubMed1993

Purification and characterization of Pseudomonas fluorescens SIK W1 lipase expressed in Escherichia coli.

Pseudomonas fluorescens SIK W1 lipase was expressed as a form of inclusion bodies in Escherichia coli, which was equivalent to 46% of total cell protein. The inclusion bodies isolated from other cell components were solubilized in the buffer containing 8 M urea and then refolded by diluting urea. The lipase with active conformation was purified by hydrophobic interaction chromatography, gel filtration, anion-exchange chromatography and hydroxyapatite chromatography from the refolded sample. By these purification steps, a single band for active lipase was detected on non-reducing SDS-PAGE and 10-fold purification was attained on the basis of specific activity. Specific activity of the purified lipase toward olive oil emulsion was found to be 7395 units per mg protein. The optimum pH and temperature of the lipase were pH 8.5 and 45-55 degrees C, respectively. The lipase showed higher lipolytic activity toward tricaproin (C6) and tricaprylin (C8) among the triacylglycerols examined and preferentially hydrolyzed ester bond of 1- and 3-position of triolein. Lipase activity was greatly increased by approx. 6-fold and stability for pH was shifted to alkaline pH by Ca2+ ion. The lipase was inhibited by Hg2+, Ag2+, p-chloromercuribenzoate, diethylpyrocarbonate and sodium dodecyl sulfate.

Amino Acid Sequence↗

PubMed1997

Partial purification and characterization of manganese-oxidizing factors of Pseudomonas fluorescens GB-1.

The Mn(2+)-oxidizing bacterium Pseudomonas fluorescens GB-1 deposits Mn oxide around the cell. During growth of a culture, the Mn(2+)-oxidizing activity of the cells first appeared in the early stationary growth phase. It depended on the O2 concentration in the culture during the late logarithmic growth phase. Maximal activity was observed at an oxygen concentration of 26% saturation. The activity could be recovered in cell extracts and was proportional to the protein concentration in the cell extracts. The specific activity was increased 125-fold by ammonium sulfate precipitation followed by reversed-phase and gel filtration column chromatographies. The activity of the partly purified Mn(2+)-oxidizing preparation had a pH optimum of circa 7 and a temperature optimum of 35 degrees C and was lost by heating. The Mn(2+)-oxidizing activity was sensitive to NaN3 and HgCl2. It was inhibited by KCN, EDTA, Tris, and o-phenanthroline. Although most data indicated the involvement of protein in Mn2+ oxidation, the activity was slightly stimulated by sodium dodecyl sulfate at a low concentration and by treatment with pronase and V8 protease. By polyacrylamide gel electrophoresis, two Mn(2+)-oxidizing factors with estimated molecular weights of 180,000 and 250,000 were detected.

Bacterial Proteins↗

PubMed1995

Mechanisms of biodegradation of metal-citrate complexes by Pseudomonas fluorescens.

Biodegradation of metal-citrate complexes by Pseudomonas fluorescens depends on the nature of the complex formed between the metal and citric acid. Bidentate Fe(III)-, Ni-, and Zn-citrate complexes were readily biodegraded, but the tridentate Cd- and Cu-citrate, and U-citrate complexes were not. The biodegradation of Ni- and Zn-citrate commenced after an initial lag period; the former showed only partial (70%) degradation, whereas the latter was completely degraded. Uptake studies with 14C-labeled citric acid and metal-citrate complexes showed that cells grown in medium containing citric acid transported free citric acid at the rate of 28 nmol min-1 and Fe(III)-citrate at the rate of 12.6 nmol min-1 but not Cd-, Cu-, Ni-, U-, and Zn-citrate complexes. However, cells grown in medium containing Ni- or Zn-citrate transported both Ni- and Zn-citrate, suggesting the involvement of a common, inducible transport factor. Cell extracts degraded Fe(III)-, Ni-, U-, and Zn-citrate complexes in the following order: The cell extract did not degrade Cd- or Cu-citrate complexes. These results show that the biodegradation of the U-citrate complex was limited by the lack of transport inside the cell and that the tridentate Cd- and Cu-citrate complexes were neither transported inside the cell nor metabolized by the bacterium.

Adaptation, Physiological↗

PubMed2006

Characterization of the substrate specificity of PhlD, a type III polyketide synthase from Pseudomonas fluorescens.

PhlD, a type III polyketide synthase from Pseudomonas fluorescens, catalyzes the synthesis of phloroglucinol from three molecules of malonyl-CoA. Kinetic analysis by direct measurement of the appearance of the CoASH product (k(cat) = 24 +/- 4 min(-1) and Km = 13 +/- 1 microM) gave a k(cat) value more than an order of magnitude higher than that of any other known type III polyketide synthase. PhlD exhibits broad substrate specificity, accepting C4-C12 aliphatic acyl-CoAs and phenylacetyl-CoA as the starters to form C6-polyoxoalkylated alpha-pyrones from sequential condensation with malonyl-CoA. Interestingly, when primed with long chain acyl-CoAs, PhlD catalyzed extra polyketide elongation to form up to heptaketide products. A homology structural model of PhlD showed the presence of a buried tunnel extending out from the active site to assist the binding of long chain acyl-CoAs. To probe the structural basis for the unusual ability of PhlD to accept long chain acyl-CoAs, both site-directed mutagenesis and saturation mutagenesis were carried out on key residues lining the tunnel. Three mutations, M21I, H24V, and L59M, were found to significantly reduce the reactivity of PhlD with lauroyl-CoA while still retaining its physiological activity to synthesize phloroglucinol. Our homology modeling and mutational studies indicated that even subtle changes in the tunnel volume could affect the ability of PhlD to accept long chain acyl-CoAs. This suggested novel strategies for combinatorial biosynthesis of unnatural pharmaceutically important polyketides.

Bacterial Proteins↗

PubMed2002

Phenotypic selection and phase variation occur during alfalfa root colonization by Pseudomonas fluorescens F113.

During colonization of the alfalfa rhizosphere, Pseudomonas fluorescens F113 undergoes phenotypic variation, resulting in the appearance of colonies with different morphology. Among phenotypic variants, three isolates, C, F, and S were selected, with the C variant showing colony morphology identical to that of the inoculated wild-type strain and F and S having a translucent and diffuse morphology. Phenotypic variants F and S were shown to preferentially colonize distal parts of the roots and showed alterations in motility, swimming faster than the C variant and swarming under conditions that did not allow swarming of the C variant. The motility behavior correlated with overproduction of the fliC-encoded protein flagellin but not with hyperflagellation. Flagella of the F and S variants were several times longer than those of the C variant, and overproduction of flagellin was regulated at the transcriptional level. Variant F showed alterations in traits that have been shown to be important for rhizosphere colonization, such as siderophore, cyanide, and exoprotease production, and these phenotypes were complemented by a cloned gacA. Sequence analysis of the gacA alelle in variant F suggested selection of the phenotype in the rhizosphere. Variant F was also affected in other phenotypes, such as lipopolysaccharide structure and flocculation in unshaken liquid medium, which were not complemented by the gacA or gacS gene. Mutation of the F113 sss gene, encoding a site-specific recombinase, showed that most of the phenotypic variation was due to the activity of this recombinase, indicating that phase variation occurs during rhizosphere colonization.

Alleles↗