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PubMed1994

Alternative routes of enzymic cyanide metabolism in Pseudomonas fluorescens NCIMB 11764.

Cell-free extracts from Pseudomonas fluorescens NCIMB 11764 catalysed the degradation of cyanide into products that included CO2, formic acid, formamide and ammonia. Cyanide-degrading activity (CDA) was localized to cytosolic cell fractions and was observed at substrate concentrations as high as 100 mM (2600 mg CN-I-1). At least two different CDAs could be distinguished by: (i) the determination of reaction product stoichiometries, (ii) requirements for NADH and oxygen, and (iii) kinetic analysis. The first activity produced CO2 and NH3 as reaction products, was dependent on oxygen and NADH for activity, and displayed an apparent Km for cyanide of 1.2 mM. The second activity generated formic acid (and NH3) plus formamide as reaction products, was oxygen independent, and had an apparent Km of 12 mM for cyanide. The first enzymic activity was identified as cyanide oxygenase as previously described [Harris, R. E. & Knowles, C. J. (1983) FEMS Microbiol Lett 20, 337-341] whereas the second activity is believed to consist of two enzymes, a cyanide nitrilase (dihydratase) and hydratase (EC 4.2.1.66). In addition to these enzymes, cyanide-grown cells were also induced for formate dehydrogenase (EC 1.2.1.2) thereby providing a means of recycling NADH utilized by cyanide oxygenase. A mutant strain having lost the ability to grow on cyanide as a nitrogen source was isolated and shown to be defective in cyanide oxygenase, but not the cyanide nitrilase/hydratase enzymes. This finding together with results showing that the substrate affinity of cyanide oxygenase was tenfold greater than for the nitrilase/hydratase enzymes, indicates that it is this enzyme that is most important in cyanide assimilation.(ABSTRACT TRUNCATED AT 250 WORDS)

Ammonia↗
PubMed1995

Amplification of the housekeeping sigma factor in Pseudomonas fluorescens CHA0 enhances antibiotic production and improves biocontrol abilities.

Pseudomonas fluorescens CHA0 produces a variety of secondary metabolites, in particular the antibiotics pyoluteorin and 2,4-diacetylphloroglucinol, and protects various plants from diseases caused by soilborne pathogenic fungi. The rpoD gene encoding the housekeeping sigma factor sigma 70 of P. fluorescens was sequenced. The deduced RpoD protein showed 83% identity with RpoD of Pseudomonas aeruginosa and 67% identity with RpoD of Escherichia coli. Attempts to inactivate the single chromosomal rpoD gene of strain CHA0 were unsuccessful, indicating an essential role of this gene. When rpoD was carried by an IncP vector in strain CHA0, the production of both antibiotics was increased severalfold and, in parallel, protection of cucumber against disease caused by Pythium ultimum was improved, in comparison with strain CHA0.

Amino Acid Sequence↗
PubMed2004

Structure and function of the phenazine biosynthetic protein PhzF from Pseudomonas fluorescens.

Phenazines produced by Pseudomonas and Streptomyces spp. are heterocyclic nitrogen-containing metabolites with antibiotic, antitumor, and antiparasitic activity. The antibiotic properties of pyocyanin, produced by Pseudomonas aeruginosa, were recognized in the 1890s, although this blue phenazine is now known to be a virulence factor in human disease. Despite their biological significance, the biosynthesis of phenazines is not fully understood. Here we present structural and functional studies of PhzF, an enzyme essential for phenazine synthesis in Pseudomonas spp. PhzF shares topology with diaminopimelate epimerase DapF but lacks the same catalytic residues. The structure of PhzF in complex with its substrate, trans-2,3-dihydro-3-hydroxyanthranilic acid, suggests that it is an isomerase using the conserved glutamate E45 to abstract a proton from C3 of the substrate. The proton is returned to C1 of the substrate after rearrangement of the double-bond system, yielding an enol that converts to the corresponding ketone. PhzF is a dimer that may be bifunctional, providing a shielded cavity for ketone dimerization via double Schiff-base formation to produce the phenazine scaffold. Our proposed mechanism is supported by mass and NMR spectroscopy. The results are discussed in the context of related structures and protein sequences of unknown biochemical function.

Bacterial Proteins↗
PubMed2001

Characterization of algG encoding C5-epimerase in the alginate biosynthetic gene cluster of Pseudomonas fluorescens.

The organization of the alginate gene cluster in Pseudomonas fluorescens was characterized. A bank of genomic DNA from P. fluorescens was mobilized to a strain of Pseudomonas aeruginosa with a transposon insertion (algJ::Tn501) in the alginate biosynthetic operon that rendered it non-mucoid. Phenotypic complementation in this heterologous host was observed, and a complementing clone containing 32 kb of P. fluorescens DNA was obtained. Southern hybridization studies showed that genes involved in alginate biosynthesis (e.g. algD, algG, and algA) were approximately in the same order and position as in P. aeruginosa. When the clone was mobilized to a P. aeruginosa algG mutant that produced alginate as polymannuronate due to its C5-epimerase defect, complementation was observed and the alginate from the recombinant strain contained L-guluronate as determined by proton nuclear magnetic resonance spectroscopy. A sequence analysis of the P. fluorescens DNA containing algG revealed sequences similar to P. aeruginosa algG that were also flanked by algE- and algX-like sequences. The predicted AlgG amino acid sequence of P. fluorescens was 67% identical (80% similar) to P. aeruginosa AlgG and 60% identical (76% similar) to Azotobacter vinelandii AlgG. As in P. aeruginosa, AlgG from P. fluorescens appeared to have a signal sequence that would localize it to the periplasm where AlgG presumably acts as a C5-epimerase at the polymer level. Non-polar algG knockout mutants of P. fluorescens were defective in alginate production, suggesting a potential role for this protein in polymer formation.

Alginates↗
PubMed2002

Cloning, functional expression and biochemical characterization of a stereoselective alcohol dehydrogenase from Pseudomonas fluorescens DSM50106.

Sequencing of a genomic library prepared from Pseudomonas fluorescens DSM 50106 identified an orf showing 29% identity to a C alpha-dehydrogenase of Pseudomonas paucimobilis and high homology to several sequences with unknown functions derived from genome projects. The corresponding gene adhF1 encodes a dehydrogenase of 296 amino acids with a calculated molecular mass of 31.997 kDa. The gene was functionally expressed in E. coli using a rhamnose inducible expression system. The resulting recombinant enzyme was active in the pH range 6-10 (best pH 8) and at 5-25 degrees C. This dehydrogenase converts cyclic ketones to the corresponding alcohols utilizing the cofactor NADH. The highest activity was found for cyclohexanone. The enzyme also exhibits high stereoselectivity in the desymmetrization of the prochiral ketone acetophenone, producing optically pure ( R)-alpha-phenyl ethanol (>99%ee) at high conversion (95%).

Alcohol Dehydrogenase↗
PubMed1962

Enzyme distribution in membrane, cytoplasm, and nuclear fractions from Pseudomonas fluorescens.

Burrous, S. E. (University of Illinois, Urbana) and W. A. Wood. Enzyme distribution in membrane, cytoplasm, and nuclear fractions from Pseudomonas fluorescens. J. Bacteriol. 84:364-369. 1962.-A procedure is described for fractionation of Pseudomonas fluorescens A3.12 by controlled rupture of spheroplasts and fractional centrifugations to yield cytoplasm, membrane, and deoxyribonucleic acid (DNA) fractions with low cross contamination and little loss of enzymatic activity. Assays for the content of a number of oxidative and nonoxidative enzymes revealed that: (i) little activity is present in the DNA fraction; (ii) only reduced diphosphopyridine nucleotide oxidase is found exclusively in the membranes; (iii) phosphohexosisomerase and probably transketolase and glucose-6-phosphate dehydrogenase are found exclusively in the cytoplasm; and (iv) the remaining five enzymes studied were found in both fractions. In contrast to earlier reports of gluconic dehydrogenase being membrane-bound and 2-keto-3-deoxy-6-phosphogluconate aldolase being soluble in sonic extracts, this procedure produced partly soluble gluconic dehydrogenase and partly membrane-associated 2-keto-3-deoxy-6-phosphogluconate aldolase.

Aldehyde-Lyases↗
PubMed2003

Identification and characterization of the emhABC efflux system for polycyclic aromatic hydrocarbons in Pseudomonas fluorescens cLP6a.

The hydrocarbon-degrading environmental isolate Pseudomonas fluorescens LP6a possesses an active efflux mechanism for the polycyclic aromatic hydrocarbons phenanthrene, anthracene, and fluoranthene but not for naphthalene or toluene. PCR was used to detect efflux pump genes belonging to the resistance-nodulation-cell division (RND) superfamily in a plasmid-cured derivative, P. fluorescens cLP6a, which is unable to metabolize hydrocarbons. One RND pump, whose gene was identified in P. fluorescens cLP6a and was designated emhB, showed homology to the multidrug and solvent efflux pumps in Pseudomonas aeruginosa and Pseudomonas putida. The emhB gene is located in a gene cluster with the emhA and emhC genes, which encode the membrane fusion protein and outer membrane protein components of the efflux system, respectively. Disruption of emhB by insertion of an antibiotic resistance cassette demonstrated that the corresponding gene product was responsible for the efflux of polycyclic aromatic hydrocarbons. The emhB gene disruption did not affect the resistance of P. fluorescens cLP6a to tetracycline, erythromycin, trimethoprim, or streptomycin, but it did decrease resistance to chloramphenicol and nalidixic acid, indicating that the EmhABC system also functions in the efflux of these compounds and has an unusual selectivity. Phenanthrene efflux was observed in P. aeruginosa, P. putida, and Burkholderia cepacia but not in Azotobacter vinelandii. Polycyclic aromatic hydrocarbons represent a new class of nontoxic, highly hydrophobic compounds that are substrates of RND efflux systems, and the EmhABC system in P. fluorescens cLP6a has a narrow substrate range for these hydrocarbons and certain antibiotics.

Anthracenes↗
PubMed2000

Effect of psychrotrophic bacteria and of an isolated protease from Pseudomonas fluorescens M3/6 on the plasmin system of fresh milk.

In the present study, we investigated the effect of Pseudomonas spp. growth on the plasmin enzymatic system in casein and whey fractions of fresh milk. Two bacterial strains, Pseudomonas spp. SRM28A and Pseudomonas fluorescens M3/6, were inoculated at a level of approximately 10(3) cfu/ml into fresh milk and incubated at 7 degrees C for 3 d. Bacterial counts were approximately 10(8) cfu/ml by d 3. Samples collected every 24 h were treated to separate the casein from the whey fraction. Casein and whey fractions were subjected to electrophoresis to visualize protein breakdown and plasmin activity and to colorimetric assays to quantify plasmin-related activities. With psychrotrophic bacterial growth, plasmin levels in casein fractions decreased significantly and in whey fractions increased then decreased significantly. Fresh milk results were similar for the two strains and were similar to earlier results with reconstituted nonfat dry milk. A transmission electron microscopy study by immunocytochemistry showed the presence of plasmin in casein micelles and its disappearance upon microbial growth in the milk. We hypothesized that extracellular microbial proteases produced by psychrotrophic microorganisms are responsible for this effect. To confirm this, an extracellular bacterial protease was isolated from Pseudomonas fluorescens M3/6 by ammonium sulfate fractionation and ion-exchange chromatography and incubated with fresh milk. Milk samples analyzed during incubation with the protease had significantly increased plasmin and plasminogen activities in the whey fraction within 5 h of incubation, while differences in activities in the casein fraction occurred at time 7.5 h for plasmin activity and 10 h of incubation for plasminogen activity. These quantitative data were supported by plasmin activity as visualized by casein-SDS-PAGE. These results suggest that growth of the Pseudomonas strains in fresh milk, and particularly their production of extracellular proteases, may be a causative factor in the release of plasmin from the casein micelle. Such plasmin release could affect the quality of cheeses and other food products that utilize dairy ingredients.

Animals↗
PubMed2001

A strain of Pseudomonas fluorescens with two lipase-encoding genes, one of which possibly encodes cytoplasmic lipolytic activity.

AIMS: A lipase-encoding gene (lipA) from a psychrotrophic strain of Pseudomonas fluorescens C9 has previously been characterized. It was also shown that when this gene was insertionally-inactivated, lipase activity was retained, suggesting that a second lipase may be present in this strain. The aim of this study was to determine whether this was the case. METHODS AND RESULTS: Using molecular cloning, chromosomal mutagenesis and enzymatic analysis, the presence of a second lipase-encoding gene (lipB) has been confirmed. The molecular weights of the putative products of lipA and lipB are 33 and 64.5 kDa, respectively, and their sequences are quite dissimilar (< 10% sequence identity). The lipB gene encodes a secreted lipase and is solely responsible for the 'lipolytic phenotype' of Ps. fluorescens C9. Expression of the lipA gene can be detected when expressed using an expression vector, but activity was only detected intracellularly in Ps. fluorescens C9, and not in the culture medium. CONCLUSION: Pseudomonas fluorescens C9 contains two dissimilar lipases. One (LipB) is secreted and responsible for the lipolytic phenotype; the evidence suggests that the other (LipA) could be intracellular, but it could be secreted and not detectable. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteria may contain more than one lipase activity. Ascribing phenotypes to particular enzymes therefore requires mutational analysis. The notion of an intracellular lipase activity is novel, and, if further substantiated, begs the question as to its normal substrate and physiological role.

Bacterial Proteins↗
PubMed1993

A sandwich enzyme-linked immunosorbent assay (ELISA) for detection of Pseudomonas fluorescens and related psychrotrophic bacteria in refrigerated milk.

A sandwich ELISA (enzyme-linked immunosorbent assay) was developed for detection of Pseudomonas fluorescens and related psychrotrophic bacteria in refrigerated milk. Polyclonal antibodies were raised in rabbits against protein F from the cell envelope of Pseudomonas fluorescens AH-70. The anti-protein F antibodies (anti-PF) bound to the wells of a microtitre plate were used to capture this protein from the micro-organisms on milk samples. Further immunorecognition of the captured antigen was attained with the same anti-PF antibodies conjugated to biotin. ExtrAvidin-peroxidase was used to detect the biotinylated antibodies bound to their specific antigens. Subsequent enzymic conversion of substrate gave clear absorbance differences when assaying milk samples containing Ps. fluorescens strains of different origin as well as related psychrotropic micro-organisms.

Animals↗
PubMed1995

Iron-responsive gene expression in Pseudomonas fluorescens M114: cloning and characterization of a transcription-activating factor, PbrA.

In response to iron limitation. Pseudomonas fluorescens M114 induces a number of genes including an iron-scavenging siderophore termed pseudobactin M114, its cognate receptor, PbuA, and a casein protease. A Tn5lacZ-induced mutant (M114FA1) was isolated that exhibits a pleiotropic phenotype and lacks the ability to express these iron-regulated genes. A cosmid clone was identified which complements this mutation. This clone is capable of activating a number of iron-regulated promoter fusion constructs from P. fluorescens M114 and Pseudomonas putida WCS358 and can also promote expression of these fusions in Escherichia coli. A series of insertion mutants was constructed by homologous recombination which were unable to transcribe the promoter fusions. DNA sequence analysis of the complementing region identified one open reading frame (ORF) termed pbrA (pseudobactin regulation activation) and the deduced amino acid sequence shows domains with significant homology to a number of ECF (extracytoplasmic function) transcriptional regulators of the sigma 70 sigma factor family, including fecl required for expression of the ferric dicitrate outer-membrane receptor protein of E. coli. Sequences upstream of the pbrA gene suggest that transcription of pbrA may also be iron regulated.

Amino Acid Sequence↗
PubMed2004

Trichoderma harzianum enhances the production of nematicidal compounds in vitro and improves biocontrol of Meloidogyne javanica by Pseudomonas fluorescens in tomato.

AIMS: To determine the influence of soil-borne fungus Trichoderma harzianum on the biocontrol performance of Pseudomonas fluorescens strain CHA0 and its 2,4-diacetylphloroglucinol (DAPG) overproducing derivative CHA0/pME3424 against Meloidogyne javanica. METHODS AND RESULTS: Amendment of the culture filtrate (CF) or methanol extract of the CF of a T. harzianum strain Th6 to P. fluorescens growth medium enhanced the production of nematicidal compound(s) by bacterial inoculants in vitro. In addition, bacteria overwhelmingly expressed phl'-'lacZ reporter gene when the medium was amended with CF of T. harzianum. Pseudomonas fluorescens and T. harzianum applied together in unsterilized sandy loam soil caused greater reduction in nematode population densities in tomato roots. CONCLUSIONS: Trichoderma harzianum improves root-knot nematode biocontrol by the antagonistic rhizobacterium P. fluorescens both in vitro and under glasshouse conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: The synergistic effect of T. harzianum on the production of nematicidal compound(s) critical in biocontrol may improve the efficacy of biocontrol bacteria against plant-parasitic nematodes. Considering the inconsistent performance of the biocontrol agents under field conditions, application of a mixture of compatible T. harzianum and P. fluorescens would more closely mimic the natural situation and might broaden the spectrum of biocontrol activity with enhanced efficacy and reliability of control.

Animals↗
PubMed1996

Sterol peroxidation by Pseudomonas fluorescens cholesterol oxidase.

Cholesterol is oxidized by commercially available Pseudomonas fluorescens cholesterol oxidase to 6 beta-hydroperoxycholest-4-en-3-one as the initial product, with none of the expected produce, cholest-4-en-3-one, formed. The transformation indicates that P. fluorescens cholesterol oxidase also acts as a flavoprotein dioxygenase.

Cholesterol↗
PubMedDate not supplied

[Serologic characteristics of Pseudomonas fluorescens strain 7769, isolated from rye].

Serological properties of Pseudomonas fluorescens 7769, isolated from the damaged tissues of rye have been studied. It has been shown that P. fluorescens 7769 was serologically related but not identical as to antigenic composition with the type strain P. fluorescens 4125 of biovar 1, to which it belonged and closely related to the strain P. fluorescens 1371, isolated from wheat. Availability of weakly expressed common antigens of P. fluorescens 7769 with the agent of the disease of grain cultures P. syringae pv. atrofaciens has been established.

Antigens, Bacterial↗
PubMedDate not supplied

The pyoverdin of Pseudomonas fluorescens G173, a novel structural type accompanied by unexpected natural derivatives of the corresponding ferribactin.

The siderophores produced by Pseudomonas fluorescens G173 are unusual in several respects. So far all pyoverdins with a C-terminal cyclopeptidic substructure have in common that the epsilon-amino group of an in-chain Lys is bound amidically to the carboxyl group of a C-terminal Ser or Thr and that N5-formyl-N5-hydroxy Orn (FoOHOrn) is the next amino acid after Lys. FoOHOrn may (cyclotetrapeptidic structures) be or may not (cyclotripeptidic structures) be followed by a further amino acid. In the pyoverdin described here Orn instead of Lys is the amino acid forming the cycle, FoOHOrn is replaced by AcOHOrn which does not follow the branching Orn but is the penultimate amino acid and finally the last amino acid is Asp. The producing strain which had been classified as Pseudomonas fluorescens may well be a new species. Pyoverdins are frequently accompanied by ferribactins which are considered to be their biogenetic precursors. They always have the same amino acid chain as the co-occurring pyoverdins but the pyoverdin chromophore is replaced by a condensation product of L-Dab and D-Tyr with the amino group of Tyr bound to the gamma-carboxyl group of Glu. A ferribactin having these structural characteristics is produced by the investigated strain, but it is accompanied by derivatives where the alpha-amino group of Glu is partially or completely transformed into a hydroxamic acid by substitution with a hydroxyl and/or acetyl group.

Amino Acids↗
PubMed1963

SOME EFFECTS OF CARBON SOURCE, AERATION, AND TEMPERATURE ON GROWTH OF A PSYCHROPHILIC STRAIN OF PSEUDOMONAS FLUORESCENS.

Olsen, Ronald H. (University of Minnesota, St. Paul) and James J. Jezeski. Some effects of carbon source, aeration, and temperature on growth of a psychrophilic strain of Pseudomonas fluorescens. J. Bacteriol. 86:429-433. 1963.-A psychrophilic strain of Pseudomonas fluorescens was grown in stationary and aerated culture at 4, 10, 15, 20, 25, and 32 C. Generation times were determined for cultures growing in media containing glucose, monosodium citrate, or Casamino Acids as the primary carbon and energy source. The results indicated that growth on Casamino Acids with aeration contributed to significantly shorter generation times at low temperatures when compared with other media systems. Furthermore, temperature coefficients (Q(10) values) calculated from growth-velocity data showed that the effects of temperature on growth rate are influenced by the carbon source in the growth medium and by aeration.

Amino Acids↗
PubMed1992

Pseudomonas fluorescens subsp. cellulosa: an alternative model for bacterial cellulase.

Pseudomonas fluorescens subsp. cellulosa, a Gram-negative soil bacterium, can utilize crystalline cellulose or xylan as main sources of carbon and energy. Synthesis of endoglucanases and xylanases is induced by Avicel, filter paper, carboxymethylcellulose or xylan and is repressed by cellobiose, glucose or xylose. These enzymes are secreted into the culture supernatant fluid and do not form aggregates or associate with the cell surface. Cells of Ps. fluorescens subsp. cellulosa do not adhere to cellulose. In cultures containing Avicel or filter paper, a significant proportion of the secreted cellulase and xylanase activities becomes tightly bound to the insoluble cellulose. Western blotting has revealed that endoglucanase B, xylanase A and a cellodextrinase encoded by genes previously isolated from Ps. fluorescens subsp. cellulosa and expressed in Escherichia coli, are synthesized by the pseudomonad under a variety of conditions. These enzymes appear to be post-translationally modified, probably through glycosylation. Overall, it appears that the cellulase/hemicellulase system of Ps. fluorescens subsp. cellulosa differs from the model established for celluloytic anaerobes such as Clostridium thermocellum.

Blotting, Western↗