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PubMed1991

Indole-3-acetic acid (IAA) synthesis in the biocontrol strain CHA0 of Pseudomonas fluorescens: role of tryptophan side chain oxidase.

Pseudomonas fluorescens strain CHA0 is an effective biocontrol agent against soil-borne fungal plant pathogens. In this study, indole-3-acetic acid (IAA) biosynthesis in strain CHA0 was investigated. Two key enzyme activities were found to be involved: tryptophan side chain oxidase (TSO) and tryptophan transaminase. TSO was induced in the stationary growth phase. By fractionation of a cell extract of strain CHA0 on DEAE-Sepharose, two distinct peaks of constitutive tryptophan transaminase activity were detected. A pathway leading from tryptophan to IAA via indole-3-acetamide, which occurs in Pseudomonas syringae subsp. savastanoi, was not present in strain CHA0. IAA synthesis accounted for less than or equal to 1.5% of exogenous tryptophan consumed by resting cells of strain CHA0, indicating that the bulk of tryptophan was catabolized via yet another pathway involving anthranilic acid as an intermediate. Strain CHA750, a mutant lacking TSO activity, was obtained after Tn5 mutagenesis of strain CHA0. In liquid cultures (pH 6.8) supplemented with 10 mM-L-tryptophan, growing cells of strains CHA0 and CHA750 synthesized the same amount of IAA, presumably using the tryptophan transaminase pathway. In contrast, resting cells of strain CHA750 produced five times less IAA in a buffer (pH 6.0) containing 1 mM-L-tryptophan than did resting cells of the wild-type, illustrating the major contribution of TSO to IAA synthesis under these conditions. In artificial soils at pH approximately 7 or pH approximately 6, both strains had similar abilities to suppress take-all disease of wheat or black root rot of tobacco. This suggests that TSO-dependent IAA synthesis is not essential for disease suppression.

Bacterial Proteins↗

PubMed1992

Detection of a novel carbon-phosphorus bond cleavage activity in cell-free extracts of an environmental Pseudomonas fluorescens isolate.

Cell-free extracts of Pseudomonas fluorescens strain 23F catalyzed the hydrolysis of phosphonoacetate to acetate and inorganic phosphate; the products were detected in almost equimolar quantities. The stable in vitro activity responsible was distinct from phosphonoacetaldehyde hydrolase and appears to represent a novel mode of carbon-phosphorus bond cleavage.

Acetates↗

PubMed1991

Relative importance of fluorescent siderophores and other factors in biological control of Gaeumannomyces graminis var. tritici by Pseudomonas fluorescens 2-79 and M4-80R.

Pseudomonas fluorescens 2-79 suppresses take-all, a major root disease of wheat caused by Gaeumannomyces graminis var. tritici. The bacteria produce an antibiotic, phenazine-1-carboxylic acid (PCA), and a fluorescent pyoverdin siderophore. Previous studies have established that PCA has an important role in the biological control of take-all but that antibiotic production does not account fully for the suppressiveness of the strain. To define the role of the pyoverdin siderophore more precisely, mutants deficient in production of the antibiotic, the siderophore, or both factors were constructed and compared with the parental strain for control of take-all on wheat roots. In all cases, strains that produced PCA were more suppressive than those that did not, and pyoverdin-deficient mutant derivatives controlled take-all as effectively as their respective fluorescent parental strains. Thus, the phenazine antibiotic was the dominant factor in disease suppression and the fluorescent siderophore had little or no role. The siderophore also was of minor importance in a second strain, P. fluorescens M4-80R, that does not produce PCA. Strains 2-79 and M4-80R both produced substances distinct from the pyoverdin siderophore that were responsible for fungal inhibition in vitro under iron limitation, but these substances also had, at most, a minor role in disease suppression in situ.

Antifungal Agents↗

PubMed2004

Adhesion of Pseudomonas fluorescens on magnetic surfaces.

The adhesion of Pseudomonas fluorescens (ATCC 700830) to perpendicularly polarized magnetic surfaces was recently discovered. The findings have found that the magnetic free surfaces from different magnetic polarities have different profound effects on the P. fluorescens bacterial adhesion to its surfaces. These phenomena can be explained by the surface magnetic effect, which was found to affect the surface free energy. An in situ experiment, by contrast microscopy and under static conditions, was conducted to determine the influence of magnetic surfaces, that are polarized under different external magnetizing field strengths, on bacterial adhesion. The effect of different magnetic polarities on the surface free energy has also been investigated.

Bacterial Adhesion↗

PubMed1994

Thermal resistance of partially purified proteinase of Pseudomonas fluorescens P-26.

Heat resistance of the Pseudomonas fluorescens P-26 proteinase in terms of D-value was studied in whole milk, skim milk, whey and 0.05 mol l-1 phosphate buffer at 72.5, 130, 135, 140, 145 and 150 degrees C subsequent to its partial purification through (NH4)2 SO4 precipitation (45-65% saturation) and solvent fractionation with 1.0 to 2.0 volumes of isopropanol. The D-value was maximum for the proteinase at all temperatures when determined in whole milk (D150 = 0.088).

Ammonium Sulfate↗

PubMed1991

Preliminary investigation of crystals of the neutral lipase from Pseudomonas fluorescens.

The neutral lipase from the bacteria Pseudomonas fluorescens, marketed under the trade name LpL-200S, has been crystallized in a form suitable for X-ray diffraction analysis from 35% n-propanol at pH 8.5. The crystals are monoclinic prisms and are of space group C2 with a = 91.00 A, b = 47.17 A, c = 35.21 A and beta = 121.43 degrees. There is one molecule of the protein as the asymmetric unit of the crystals. The diffraction pattern extends to at least 1.6 A resolution and the crystals are extremely robust in terms of X-ray exposure.

Crystallization↗

PubMed1997

Cloning of the phosphonoacetate hydrolase gene from Pseudomonas fluorescens 23F encoding a new type of carbon-phosphorus bond cleaving enzyme and its expression in Escherichia coli and Pseudomonas putida.

The phnA gene encoding a novel carbon-phosphorus bond cleavage enzyme, phosphonoacetate hydrolase, from Pseudomonas fluorescens 23F was cloned and expressed in Escherichia coli and Pseudomonas putida. It conferred on the latter host the ability to mineralize phosphonoacetate but on the former the ability to utilize it as sole phosphorus source only. The nucleotide and deduced amino acid sequences of the phnA gene showed no significant homology with any data bank accessions.

Alkaline Phosphatase↗

PubMed2000

The trehalose operon of Pseudomonas fluorescens ATCC 17400.

The trehalose operon of Pseudomonas fluorescens ATCC 17400 consists of treP, treA and treR. The gene treP codes for a putative enzyme II subunit of the phosphotransferase system that catalyzes the phosphorylation of trehalose together with its translocation across the cell membrane and treA encodes a putative phosphotrehalase, which hydrolyzes the incoming trehalose-6-phosphate into glucose and glucose-6-phosphate. Both genes are negatively regulated by TreR, a repressor of the FadR-GntR family of transcription regulators. The operon that is induced by trehalose present in the medium shows a high similarity both in the function of genes and in the regulation with the trehalose operon of Bacillus subtilis.

Amino Acid Sequence↗

PubMed1980

Energization of glucose transport by Pseudomonas fluorescens.

We have measured the capacity of Pseudomonas fluorescens to transport the glucose analog 2-deoxy-d-glucose and the amino acids l-alanine and alpha-aminoisobutyric acid under conditions in which the cells could generate (i) both a membrane proton motive force and high-energy phosphate compounds, (ii) a proton motive force but not high-energy phosphate compounds, and (iii) neither a proton motive force nor high-energy phosphate compounds. This was done by depleting cells of adenosine triphosphate stores by treatment with sodium arsenate and then suspending them in a phosphate-free medium, where they could generate a proton motive force but not phosphate bond energy, or in a phosphate-containing medium, where they could generate both a proton motive force and phosphate bond energy. Inclusion of the proton-conducting ionophore carbonyl cyanide-m-chlorophenyl hydrazone under either condition precluded the generation of both a proton motive force and phosphate bond energy. The amino acids l-alanine and alpha-aminoisobutyric acid were transported independently of phosphate bond energy and required only a proton motive force. 2-Deoxy-d-glucose was transported only under conditions in which phosphate bond energy could be generated. These results are consistent with the findings of others that Pseudomonas aeruginosa produces an inducible shock-sensitive glucose-binding protein and conform to the generalization that binding protein-associated transport systems are energized by phosphate bond energy.

Adenosine Triphosphate↗

PubMed1982

Isolation and general characterization of a heat-stable proteinase from Pseudomonas fluorescens aft 36.

An extracellular proteinase from Pseudomonas fluorescens, strain AFT 36, was isolated to homogeneity by chromatography on DEAE-cellulose and Sephadex G-150; a 230-fold increase in specific activity with a recovery of 53% was obtained. The enzyme was optimally active at pH 6.5 and 45 degrees C; activity declined rapidly at higher temperatures but significant activity persisted down to 4 degrees C. Activity was strongly inhibited by 10(-3) M EDTA and was partially restored by addition of Zn2+, Ca2+ or Co2+. The Km values on methylated casein and sodium caseinate were 18.2 and 7.1 mg/ml, respectively. The enzyme was very labile in phosphate buffer and in a mild salts buffer at 55 degrees C but was very stable in the latter at more than 80 degrees C.

Cations, Divalent↗

PubMed1993

Purification and some properties of proteinase from Pseudomonas fluorescens No. 33.

The extracellular proteinase from Pseudomonas fluorescens No. 33 was purified to electrophoretic homogeneity by a procedure including precipitation with HCl and (NH4)2SO4, and column chromatography. The enzyme was purified 170-fold giving a yield of 7% of the original activity. The molecular mass of the purified enzyme was 48,000 by SDS-PAGE. The optimum pH and temperature for the hydrolysis of casein were 8.0-9.8 and 30-35 degrees C respectively. The enzyme was more thermostable in synthetic milk salts solution than in 0.1 M-sodium phosphate buffer, but was heat-labile at 50 degrees C in both buffer systems. The activity was inhibited by o-phenanthroline, Hg2+, Cu2+, Fe2+ and, to a lesser extent, Ni2+. Caseins were susceptible to the proteinase, but degradation patterns were dependent on the form of the casein.

Caseins↗

PubMed1980

Evolution in Pseudomonas fluorescens.

The relationships among 93 strains of Pseudomonas fluorescens were investigated by (1) a numerical taxonomic analysis on the results of 150 phenotypic tests, (2) DNA hybridization studies using 16 reference strains, (3) quantitative microcomplement fixation studies using six reference strains with antibodies directed against the protein azurin. In general, the strains fell into distinct clusters. Assignment to these clusters on the basis of azurin immunological similarity showed 98% agreement with assignment based on DNA homology, suggesting that many genes will follow the same pattern. Of the strains that clustered on the basis of genotype (DNA, azurin) 88% also clustered on the basis of phenotype. The occasional noncongruency observed between the genotypic and phenotypic data may be due to the variable rates of phenotypic evolution. These results provide a perspective on the roles of horizontal and vertical transfer of genes in the evolution of this bacterial group.

Azurin↗

PubMed1984

Purification and some properties of intracellular esterase from Pseudomonas fluorescens.

A novel esterase was found in Pseudomonas fluorescens cells and purified to homogeneity as determined by polyacrylamide gel electrophoresis. The esterase was extracted from the cells by freeze-thawing and hypotonic treatment. Purification was achieved by ammonium sulfate precipitation, followed by successive chromatographies on DEAE-cellulose and benzylamine-agarose and then electrophoresis. The enzyme catalyzed the hydrolysis of methyl esters, such as methyl butyrate, but its hydrolyzing activity decreased with increase in the chain length of the alcohol moiety, and it did not catalyze the hydrolysis of triacylglycerols, such as triacetin. In contrast, the enzyme acted on various acyl residues in a series of methyl esters, such as dimethyl succinate, methyl methacrylate, and dimethyl malate. The optimum pH for activity of this enzyme with methyl butyrate was 7.0-8.5. The enzyme was inhibited by phenylmethylsulfonylfluoride. Its molecular weight was estimated as 48,000 by molecular sieve electrophoresis and gel filtration on Sephadex G-150.

Chromatography, DEAE-Cellulose↗

PubMed1988

Characterization and expression in Escherichia coli of an endoglucanase gene of Pseudomonas fluorescens subsp. cellulosa.

An endoglucanase gene of Pseudomonas fluorescens subsp. cellulosa present on plasmid pRUCL150 and expressed in Escherichia coli was subcloned in plasmid pBR322. Plasmid pRUCL153 contained the smallest DNA insert (2.9 kb) with endoglucanase activity. The plasmids directed the synthesis of a mostly periplasmic enzyme in E. coli and the level of enzyme activity was comparable in several strains. Analysis by non-denaturing polyacrylamide gel electrophoresis of the endoglucanase produced with various recombinant plasmids showed that it was unique. The endoglucanase gene on plasmid pRUCL153 was localized by physical mapping of independent transposon Tn5 insertions. Hence, its size was estimated to be approx. 1.3 kb. In vivo radioactive labelling of plasmid-encoded proteins using minicells, followed by denaturing polyacrylamide gel electrophoresis, allowed us to determine the size of the endoglucanase: Mr 40,000 for the precursor and Mr 38,000 for the mature enzyme. It was demonstrated that no cellulase operon, but a single gene, was cloned. The direction of transcription of the gene was determined by placing it under the control of the promoter of the lactose operon.

Cellulase↗

PubMedDate not supplied

[Asparaginase and glutaminase activity in Pseudomonas fluorescens in continuous cultivation].

The cells of Pseudomonas fluorescens AG contain two inducable asparaginase enzymes: one of them hydrolyzes only L-asparagine (asparaginase A), the other--L-asparagine, L-glutamine, and D-asparagine (asparaginase AG). In the conditions of continuous cultivation of the bacteria, aspartic and glutamic acids induce the formation of these enzymes only when the amino acids were used simultaneously as a growth-limiting factor and as a sole source of carbon and nitrogen. Both enzymes are not induced in the conditions when the growth is limited by the nitrogen of these amino acids. When the growth was limited by carbon, asparagine, aspartic and glutamic acids induce asparaginase AG more than asparaginase A. Asparagine and glutamine are better inductors than the corresponding amino acids. The activity of asparaginase and glutaminase increases with the specific growth rate of the culture. The induced synthesis of both amidases, after prolonged growth of the culture on a defined medium with glycerol, is inhibited by glycerol but not by glucose. The results are discussed from the viewpoint of regulation of amidases in these bacterial cells.

Asparaginase↗

PubMed1995

Structures of decasaccharide and tridecasaccharide tetraphosphates isolated by strong alkaline degradation of O-deacylated lipopolysaccharide of Pseudomonas fluorescens strain ATCC 49271.

Mild hydrazinolysis of Pseudomonas fluorescens strain ATCC 49271 lipopolysaccharide (LPS) followed by strong alkaline degradation and purification by anion-exchange HPLC resulted in two phosphorylated oligosaccharides (1 and 2). On the basis of compositional analysis and 1H, 13C, and 31P NMR spectroscopy, including 2D correlation spectroscopy (COSY), 2D rotating frame NOE spectroscopy (ROESY), and 2D inverse mode H-detected heteronuclear 1H-13C and 1H-31P correlation spectroscopy, the following two structures (1 and 2) could be identified [formula: see text] where Hep is L-glycero-D-manno-heptose, Kdo is 3-deoxy-D-manno-octulosonic acid, Non is 5,7-diamino-3,5,7,9-tetradeoxy-D-glycero-L-galacto-nonulosonic acid, and P is phosphate. Decasaccharide 1 and tridecasaccharide 2 represent an incomplete core and the complete core carrying one O-antigen repeating unit, respectively. Both are attached to the lipid A backbone but, due to their degradation protocol, they lack N- and O-acyl substituents, including N- and O-acetyl groups, the 5-N-acetimidoyl group of Non, the 2-N-alanyl group of GalN, and the 7-O-carbamoyl group of Hep as well as diphosphate, triphosphate, and, probably, some of the monophosphate groups that are present in the intact core oligosaccharide.

Alkalies↗

PubMed2006

Mutations in the central cavity and periplasmic domain affect efflux activity of the resistance-nodulation-division pump EmhB from Pseudomonas fluorescens cLP6a.

The EmhABC efflux system in Pseudomonas fluorescens cLP6a is homologous to the multidrug and solvent efflux systems belonging to the resistance-nodulation-division (RND) family and is responsible for polycyclic aromatic hydrocarbon transport, antibiotic resistance, and toluene efflux. To gain a better understanding of substrate transport in RND efflux pumps, the EmhB pump was subjected to mutational analysis. Mutagenesis of amino acids within the central cavity of the predicted three-dimensional structure of EmhB showed selective activity towards antibiotic substrates. An A384P/A385Y double mutant showed increased susceptibility toward rhodamine 6G compared to the wild type, and F386A and N99A single mutants showed increased susceptibility to dequalinium compared to the wild type. As well, the carboxylic acid side chain of D101, located in the central cavity region, was found to be essential for polycyclic aromatic hydrocarbon transport and resistance to all antibiotic substrates of EmhB. Phenylalanine residues located within the periplasmic pore domain were also targeted for mutagenesis, and the F325A and F281A mutations significantly impaired efflux activity for all EmhB substrates. One mutation (A206S) in the outer membrane protein docking domain increased antibiotic resistance and toluene tolerance, demonstrating the important role of this domain in transport activity. These data demonstrate the roles of the central cavity and periplasmic domains in the function of the RND efflux pump EmhB.

Anti-Bacterial Agents↗

PubMed1999

Autolysis of the proteinase from Pseudomonas fluorescens.

The gene encoding the proteinase from Pseudomonas fluorescens was cloned and sequenced in an effort to identify the cleavage sites involved in its autolysis at 50 degrees C. A single open reading frame consisting of 1449 nucleotides, encoding a protein of 482 amino acids, was found. Analysis of the N-terminal amino acid sequence of the purified proteinase indicated the presence of a prosequence consisting of 13 amino acid residues. The molecular weight of the mature protein was calculated as 48,900 based on the deduced amino acid sequence, which was consistent with that of the purified proteinase as determined by sodium dodecylsulfate-PAGE. Greater than 90% loss of proteolytic activity was observed upon heating at 50 degrees C for 2 min compared with the unheated enzyme. Incubation of the proteinase at 50 degrees C led to disappearance of the intact enzyme, as shown by sodium dodecyl sulfate-PAGE, whereas it was stable in the presence of the protease inhibitor o-phenanthroline. Autolytic fragments were fractionated by reverse-phase HPLC and subjected to N-terminal amino acid sequence analysis in an effort to determine the cleavage sites. The cleavage profile was not definitive; however, amino acid residues with small side chain groups, such as glycine or alanine, were frequently found adjacent to the cleavage sites.

Amino Acid Sequence↗