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Isolation of alginate-producing mutants of Pseudomonas fluorescens, Pseudomonas putida and Pseudomonas mendocina.

Spontaneous alginate-producing (muc) variants were isolated from strains of Pseudomonas fluorescens, P. putida and P. mendocina at a frequency of 1 in 10(8) by selecting for carbenicillin resistance. The infrared spectrum of the bacterial exopolysaccharide was typical of an acetylated alginate similar to that previously described in Azotobacter vinelandii and in mucoid variants of P. aeruginosa. Mucoid variants were not isolated from P. stutzeri, P. pseudoalcaligenes, P. testosteroni, P. diminuta, P. acidovorans, P. cepacia or P. maltophilia.

Alginates↗

Probing of Pseudomonas aeruginosa, Pseudomonas aureofaciens, Burkholderia (Pseudomonas) cepacia, Pseudomonas fluorescens, and Pseudomonas putida with the ferripyochelin receptor A gene and the synthesis of pyochelin in Pseudomonas aureofaciens, Pseudomonas fluorescens, and Pseudomonas putida.

The ferripyochelin receptor A (fptA) gene codes for the transport of the ferrisiderophore ferripyochelin in Pseudomonas aeruginosa. A P. aeruginosa fptA internal fragment was used to probe chromosomal DNA from P. aureofaciens, B. cepacia, P. fluorescens, P. putida, and five strains of P.aeruginosa. These bacteria all contained DNA that hybridized to the fptA fragment. Four of the five P. aeruginosa strains displayed marked and identical patterns, indicating a high degree of sequence similarities among these strains. DNA from the non-P. aeruginosa bacteria, in contrast, hybridized less to the fptA fragment. Pseudomonas aeruginosa and B. cepacia synthesize pyochelin. Experiments were performed to confirm P. fluorescens pyochelin synthesis and to determine if pyochelin, cepabactin or salicylic acid were made by P. aureofaciens, P. putida, and P. fluorescens. Only pyochelin was isolated and identified from P. fluorescens. P. aureofaciens and P. putida produced none of these compounds. While all of these bacteria contain chromosomal DNA that hybridized to the fptA fragment probe, pyochelin synthesis did not occur in all, indicating that fptA fragment hybridization cannot always be correlated with pyochelin biosynthesis.

Bacterial Outer Membrane Proteins↗

Transposon and spontaneous deletion mutants of plasmid-borne genes encoding polycyclic aromatic hydrocarbon degradation by a strain of Pseudomonas fluorescens.

Pseudomonas fluorescens strain LP6a, isolated from petroleum condensate-contaminated soil, utilizes the polycyclic aromatic hydrocarbons (PAHs) naphthalene, phenanthrene, anthracene and 2-methylnaphthalene as sole carbon and energy sources. The isolate also co-metabolically transforms a suite of PAHs and heterocycles including fluorene, biphenyl, acenaphthene, 1-methylnaphthalene, indole, benzothiophene, dibenzothiophene and dibenzofuran, producing a variety of oxidized metabolites. A 63 kb plasmid (pLP6a) carries genes encoding enzymes necessary for the PAH-degrading phenotype of P. fluorescens LP6a. This plasmid hybridizes to the classical naphthalene degradative plasmids NAH7 and pWW60, but has different restriction endonuclease patterns. In contrast, plasmid pLP6a failed to hybridize to plasmids isolated from several phenanthrene-utilizing strains which cannot utilize naphthalene. Plasmid pLP6a exhibits reproducible spontaneous deletions of a 38 kb region containing the degradative genes. Two gene clusters corresponding to the archetypal naphthalene degradation upper and lower pathway operons, separated by a cryptic region of 18 kb, were defined by transposon mutagenesis. Gas chromatographic-mass spectrometric analysis of metabolites accumulated by selected transposon mutants indicates that the degradative enzymes encoded by genes on pLP6a have a broad specificity permitting the oxidation of a suite of polycyclic aromatic and heterocyclic substrates.

Biodegradation, Environmental↗

Biological control of apple blue mold with Pseudomonas fluorescens.

Pseudomonas fluorescens isolate 1100-6 was evaluated as a potential biological control agent for apple blue mold caused by Penicillium expansum or Penicillium solitum. Both the wild-type isolate 1100-6 and a genetically modified derivative labeled with the gene encoding the green fluorescent protein (GFP) were compared. The P. fluorescens isolates with or without GFP equally reduced the growth of Penicillium spp. and produced large zones of inhibition in dual culture plate assays. Cell-free metabolites produced by the bacterial antagonists reduced the colony area of Penicillium isolates by 17.3% to 78.5%. The effect of iron chelate on the antagonistic potential of P. fluorescens was also studied. The use of iron chelate did not have a major effect on the antagonistic activity of P. fluorescens. With or without GFP, P. fluorescens significantly reduced the severity and incidence of apple decay by 2 P. expansum isolates after 11 d at 20 degrees C and by P. expansum and P. solitum after 25 d at 5 degrees C when the biocontrol agents were applied in wounds 24 or 48 h before challenging with Penicillium spp. Populations of P. fluorescens labeled with the GFP were determined 1, 9, 14, and 20 d after inoculation at 5 degrees C. The log CFU/mL per wound increased from 6.95 at the time of inoculation to 9.12 CFU/mL (P < 0.05) 25 d after inoculation at 5 degrees C. The GFP strain did not appear to penetrate deeply into wounds based on digital photographs taken with an inverted fluorescence microscope. These results indicate that P. fluorescens isolate 1100-6 could be an important new biological control for apple blue mold.

Green Fluorescent Proteins↗

In vivo construction of a hybrid pathway for metabolism of 4-nitrotoluene in Pseudomonas fluorescens.

Pseudomonas fluorescens 410PR grows on 4-nitrobenzoate but does not metabolize 4-nitrotoluene. The TOL pWW0 delta pm plasmid converts 4-nitrotoluene into 4-nitrobenzoate through its upper pathway, but it does not metabolize 4-nitrobenzoate. P. fluorescens 410PR(pWW0 delta pm) transconjugants were isolated and found to be able to grow on 4-nitrotoluene. This phenotype was stable after growth for at least 300 generations without any selective pressure. P. fluorescens 410PR(pWW0 delta pm) converted 4-nitrotoluene into 4-nitrobenzoate via 4-nitrobenzylalcohol and 4-nitrobenzaldehyde. 4-Nitrobenzoate was metabolized via 4-hydroxylaminobenzoate and finally yielded NH4+ and 3,4-dihydroxybenzoate, which was mineralized.

Biotransformation↗

Pseudobacteremia due to Pseudomonas fluorescens.

Pseudomonas fluorescens was recovered from 62 of 22,270 (0.26%) blood cultures, from 57 patients, over a 22-month period at a pediatric hospital. No illness was attributable to the blood culture isolate. A case-control study identified a significant correlation between the recovery of P. fluorescens in blood culture and concomitant coagulation studies (p less than 0.0001). In all cases blood for coagulation studies had been obtained at the same time as the blood culture. A review of venipuncture technique revealed that occasionally the coagulation study tubes (containing 3.8% sodium citrate) were being inoculated before blood culture bottles. P. fluorescens was subsequently isolated from coagulation tubes and from sodium citrate solutions prepared and dispensed in the hospital for use in coagulation studies. In vitro studies confirmed that sodium citrate solutions supported the growth of P. fluorescens, with preferential growth at 25 degrees C and 4 degrees C. This is the first description of P. fluorescens as a cause of pseudobacteremia. Pseudobacteremia was attributed to cross-contamination of blood cultures following inoculation of contaminated citrated collection tubes.

Adolescent↗

Glucose uptake and phosphorylation in Pseudomonas fluorescens.

Pseudomonas fluorescens ATCC 13525 and a particulate glucose oxidase (d-glucose:oxygen oxidoreductase, EC 1.1.3.4) mutant of this organism, gox-7, were examined to determine if glucose oxidation via particulate glucose oxidase is a required first step for glucose uptake. Initial [(14)C]glucose-uptake rates in parent and gox-7 cells were qualitatively similar. Initial [(14)C]glucose-uptake product analysis revealed that glucose was accumulated via active transport and was rapidly metabolized to glucose-6-phosphate and gluconate-6-phosphate in both parent and gox-7 cells. Cell extracts contained soluble adenosine 5'-triphosphate specific kinase activity for phosphorylation of glucose. Glucose uptake was induced by glucose and not gluconate, thus, establishing independent regulation of glucose transport and glucose catabolism in p. fluorescens. The results prove that glucose oxidase was not an obligatory reaction for glucose carbon permeation in P. fluorescens. A general unifying scheme for glucose utilization in the aerobic fluorescent pseudomonads is suggested for the purpose of clarifying glucose uptake in these bacteria.

Aerobiosis↗

Sequence heterogeneity of the ferripyoverdine uptake (fpvA), but not the ferric uptake regulator (fur), genes among strains of the fluorescent pseudomonads Pseudomonas aeruginosa, Pseudomonas aureofaciens, Pseudomonas fluorescens and Pseudomonas putida.

Pseudomonas aeruginosa, Pseudomonas aureofaciens, Pseudomonas fluorescens and Pseudomonas putida are of importance to medicine, agriculture and biocycling. These microbes acquire ferric ion via the use of the siderophores pyochelin and the family known as the pyoverdines or pseudobactins. The ferric uptake regulator (fur) gene is responsible, at least in part, for the regulation of siderophore synthesis and uptake in P. aeruginosa. To determine whether the organisms contain single or multiple homologues of the siderophore-related genes fpvA (ferripyoverdine uptake) and fur, and whether these homologues displayed sequence heterogeneity, their chromosomal DNAs were probed with fur and fpvA sequences. As a representative of a non-fluorescent pseudomonad, the bacterium Burkholderia (Pseudomonas) cepacia was also examined. The pseudomonads all contained fpvA- and fur-like homologues, and heterogeneity was observed among the different species. The presence of two or more fpvA-like genes is indicated in all of the fluorescent pseudomonads surveyed. In contrast, B. cepacia DNA either did not hybridize to these probes, or did so only very weakly, suggesting that fur- and fpvA-like homologues are either absent or significantly different in B. cepacia compared to the fluorescent pseudomonads examined.

Bacterial Outer Membrane Proteins↗

Isolation and identification of N-mercapto-4-formylcarbostyril, an antibiotic produced by Pseudomonas fluorescens.

Pseudomonas fluorescens strain G308 isolated from barley leaves produces a novel antibiotic substance that was purified by preparative TLC and HPLC and identified as N-mercapto-4-formylcarbostyril (Cbs) by LC/DAD, IR, LC-ES(+)/MS, LC-ES(-)/MS, GC-EI/MS, LC-HRES(+)/MS, mass isotope ratios analysis, 1H NMR and 13C NMR analysis. The purified new antibiotic compound is effective against many phytopathogenic fungi in vitro. The compound inhibited at 25 ppm spore germination and germ tube growth of the following fungi; Fusarium oxysporum f. sp. lycopersici, Fusarium culmorum, Cladosporium cucumerinum and Colletotrichum lagenarium. At concentrations up to 125 ppm, the compound did not interfere with release of zoospores from sporangia and germination of encysted zoospores of Phytophthora infestans.

Antifungal Agents↗

Maltose metabolism of Pseudomonas fluorescens.

Pseudomonas fluorescens W uses maltose exclusively by hydrolyzing it to glucose via an inducible alpha-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20). No evidence for phosphorolytic cleavage or oxidation to maltobionic acid was found in this organism. The alpha-glucosidase was totally intracellular and was most active at pH of 7.0. Induction occurred when cells were incubated with maltotriose or maltose. Induction was rapid and easily detectable within the first 5 min after the addition of the inducer. Glucose and its derivatives did not repress induction. Cells growing on DL-alanine or succinate plus maltose exhibited lower levels of alpha-glucosidase than those grown on maltose alone or maltose plus glucose. Induction required both messenger ribonucleic acid and protein synthesis.

Cell-Free System↗

CO2-fixing enzymes in Pseudomonas fluorescens.

Pseudomonas fluorescens grown on glucose or glutamate at 1 or 20 degrees C, or on acetate at 20 degrees C, as sole carbon sources, contained both pyruvate carboxylase and phosphoenolpyruvate carboxylase. Pyruvate carboxylase was insensitive to acetyl-coenzyme A and L-aspartate, and its level in cell-free extracts was markedly dependent on the carbon source for growth, the highest specific activity being attained in glucose-grown cells. Phosphoenolpyruvate carboxylase, on the other hand, although less dependent on the nature of the carbon source,showed its highest level in acetate-grown cells; the enzyme activity required acetyl-coenzyme A and was strongly inhibited by L-aspartate. The micro-organism had, in addition, a phosphoenolpyruvate carboxykinase, which showed its highest specific activity in cells grown on acetate, and a NADP-linked malate enzyme, apparently repressed by acetate and showing its highest specific activity in glutamate-grown cells.

Acetates↗

Effect of growth temperature on the lipids of Pseudomonas fluorescens.

Pseudomonas fluorescens was grown in co ntinuous culture with glucose or NH4+ as the growth-limiting substrate. The total amount of lipid and the relative proportions of neutral lipid and phospholipid did not vary with the rate or temperature of growth. The amounts of the phospholipids, which were phosphatidyl glycerol, cardiolipin and phosphatidyl ethanolamine, altered with the type of growth limitation and with the temperature and rate of growth. A precise composition of phospholipid classes is not a requirement for growth at low temperature. Fatty acid composition also varied with changes of these growth conditions at temperatures above 10 degrees C, but at lower growth temperatures the degree of saturation of the lipids was strictly controlled, and was unaffected by changes in the growth rate or the nature of the growth-limiting substrate. The degree of saturation of the lipids was not critical at the higher growth temperatures, and the variation in the degree of saturation observed at these temperatures was due in part to a decreased ability of the organism to control the fatty acid composition of its lipids at temperatures above 10 degrees C.

Cardiolipins↗

Structure and mechanism of the ThDP-dependent benzaldehyde lyase from Pseudomonas fluorescens.

Pseudomonas fluorescens is able to grow on R-benzoin as the sole carbon and energy source because it harbours the enzyme benzaldehyde lyase that cleaves the acyloin linkage using thiamine diphosphate (ThDP) as a cofactor. In the reverse reaction, this lyase catalyses the carboligation of two aldehydes with high substrate and stereospecificity. The enzyme structure was determined by X-ray diffraction at 2.6 A resolution. A structure-based comparison with other proteins showed that benzaldehyde lyase belongs to a group of closely related ThDP-dependent enzymes. The ThDP cofactors of these enzymes are fixed at their two ends in separate domains, suspending a comparatively mobile thiazolium ring between them. While the residues binding the two ends of ThDP are well conserved, the lining of the active centre pocket around the thiazolium moiety varies greatly within the group. Accounting for the known reaction chemistry, the natural substrate R-benzoin was modelled unambiguously into the active centre of the reported benzaldehyde lyase. Due to its substrate spectrum and stereospecificity, the enzyme extends the synthetic potential for carboligations appreciably.

Aldehyde-Lyases↗

Cellular response to a multiple-metal stress in Pseudomonas fluorescens.

Pseudomonas fluorescens multiplied in a minimal mineral medium supplemented with millimolar amounts of aluminum (5 mM), iron (5 mM), zinc (3 mM), calcium (2 mM) and gallium (1 mM). A slight decrease in growth rate and a 22% diminution in cellular yield were observed as compared to the control medium. Citrate, the sole source of carbon to which the test metals were complexed, was completely utilized. Although at stationary phase of growth most of the metals were immobilized in an exocellular lipid-rich residue, ultracentrifugation and dialysis studies revealed that metals were associated with phosphatidylethanolamine (PE) from early stages of growth. As growth progressed the metal content of the soluble cellular extract increased reaching an optimum at 35 h of incubation. However, no detectable amounts of metals in this cellular component were discerned at stationary phase of growth. There appeared to be no marked variation in exocellular protein and carbohydrate production in control and metal-stressed cultures. Transmission electron microscopic studies revealed metal rich bodies associated with the cytoplasm. Scanning electron microscopic analyses of the dialyzate aided in the identification of the metal-rich bodies associated with elongated structures comprised of carbon, oxygen and phosphorus. PE appeared to be an important organic constituent of the gelatinous residue.

Biotechnology↗

Identification and manipulation of soil properties to improve the biological control performance of phenazine-producing Pseudomonas fluorescens.

Pseudomonas fluorescens 2-79RN(10) protects wheat against take-all disease caused by Gaeumannomyces graminis var. tritici; however, the level of protection in the field varies from site to site. Identification of soil factors that exert the greatest influence on disease suppression is essential to improving biocontrol. In order to assess the relative importance of 28 soil properties on take-all suppression, seeds were treated with strain 2-79RN(10) (which produces phenazine-1-carboxylate [PCA(+)]) or a series of mutants with PCA(+) and PCA(-) phenotypes. Bacterized seeds were planted in 10 soils, representative of the wheat-growing region in the Pacific Northwest. Sixteen soil properties were correlated with disease suppression. Biocontrol activity of PCA(+) strains was positively correlated with ammonium-nitrogen, percent sand, soil pH, sodium (extractable and soluble), sulfate-sulfur, and zinc. In contrast, biocontrol was negatively correlated with cation-exchange capacity (CEC), exchangeable acidity, iron, manganese, percent clay, percent organic matter (OM), percent silt, total carbon, and total nitrogen. Principal component factor analysis of the 16 soil properties identified a three-component solution that accounted for 87 percent of the variance in disease rating (biocontrol). A model was identified with step-wise regression analysis (R(2) = 0.96; Cp statistic = 6.17) that included six key soil properties: ammonium-nitrogen, CEC, iron, percent silt, soil pH, and zinc. As predicted by our regression model, the biocontrol activity of 2-79RN(10) was improved by amending a soil low in Zn with 50 micro g of zinc-EDTA/g of soil. We then investigated the negative correlation of OM with disease suppression and found that addition of OM (as wheat straw) at rates typical of high-OM soils significantly reduced biocontrol activity of 2-79RN(10).

Ascomycota↗

Influence of side-chain substituents on the position of cleavage of the benzene ring by Pseudomonas fluorescens.

Pseudomonas fluorescens was grown on mineral salts media with phenol, p-hydroxybenzoic acid, p-hydroxy-phenylacetic acid, or p-hydroxy-trans-cinnamic acid as sole carbon and energy source. Each compound was first hydroxylated, ortho to the hydroxyl group on the benzene ring, to give catechol, protocatechuic acid (3,4-dihydroxy-benzoic acid), homoprotocatechuic acid (3,4-dihydroxy-phenylacetic acid), and caffeic acid (3,4-dihydroxy-trans-cinnamic acid), respectively, as the ultimate aromatic products before cleavage of the benzene nucleus. Protocatechuic acid and caffeic acid were shown to be cleaved by ortho fission, via a 3,4-oxygenase mechanism, to give beta-substituted cis, cis-muconic acids as the initial aliphatic products. However, catechol and homoprotocatechuic acid were cleaved by meta fission, by 2,3-and 4,5-oxygenases, respectively, to give alpha-hydroxy-muconic semialdehyde and alpha-hydroxy-gamma-carboxymethyl muconic semialdehyde as initial aliphatic intermediates. Caffeic acid: 3,4-oxygenase, a new oxygenase, consumes 1 mole of O(2) per mole of substrate and has an optimal pH of 7.0. The mechanism of cleavage of enzymes derepressed for substituted catechols by P. fluorescens apparently changes from ortho to meta with the increasing nephelauxetic (electron donor) effect of the side-chain substituent.

Benzene Derivatives↗

Microbial metabolism of quinoline and related compounds. II. Degradation of quinoline by Pseudomonas fluorescens 3, Pseudomonas putida 86 and Rhodococcus spec. B1.

Quinoline catabolism was investigated with different bacterial strains, able to use quinoline as sole source of carbon, nitrogen and energy. Some degradation products of quinoline were isolated from the culture fluids and identified. With Pseudomonas fluorescens and Pseudomonas putida we found 2-oxo-1,2-dihydroquinoline, 8-hydroxy-2-oxo-1,2-dihydroquinoline, 8-hydroxycoumarin and 2,3-dihydroxyphenylpropionic acid as intermediates. With a Rhodococcus strain 2-oxo-1,2-dihydroquinoline, 6-hydroxy-2-oxo-1,2-dihydroquinoline, a red meta-cleavage product and a blue fluorescent compound were isolated. The red compound was identified as 5-hydroxy-6-(3-carboxy-3-oxopropenyl)-1H-2-pyridone. From this the blue fluorescent azacoumarin 2H-pyrano-2-one-[3,2b]-5H-6-pyridone is formed by chemical decomposition. Therefore it can be considered a by-product of quinoline-degradation in Rhodococcus spec. With the present results two different degradation pathways for quinoline in different microorganisms are proposed.

Chemical Phenomena↗