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PubMed2002

Survival of the rhizosphere-competent biocontrol strain Pseudomonas fluorescens NBRI2650 in the soil and phytosphere.

Pseudomonas fluorescens NBRI2650 was isolated after screening 360 bacterial strains from the rhizosphere of chickpea (Cicer arietinum L.) grown in fungal-disease-suppressive field soil. The strain was selected because of its high rhizosphere competence and ability to inhibit the growth of Fusarium oxysporum f.sp. ciceri, Rhizoctonia bataticola, and Pythium sp. under in vitro conditions. Survival and colonization of NBRI2650 in the phytosphere of chickpea, cotton (Gossypium hirsutum L.), cucumber (Cucumis sativus L.), and tomato (Lycopersicon seculentum Mill.) were monitored using a chromosomally located rifampicin-marked mutant P. fluorescens NBRI2650R. The strain showed variable ability to invade and survive in the phytosphere of different plants. Chickpea was used as a tester plant for further work, as it was not invaded by NBRI2650R. The interaction between NBRI2650R and F oxysporum fsp. ciceri was studied by both light microscopy and scanning electron microscopy. The lysis of the fungal cell wall by NBRI2650R was clearly demonstrated. Treatment of the chickpea seeds with NBRI2650R in prerelease experiments in the greenhouse using disease-conducive field soils from Jhansi and Kanpur resulted in increased plant growth and did not result in any perturbation of the indigenous microbial community that inhabited the rhizosphere of chickpea compared with nonbacterized seeds. Direct fermentation of diluted NBRI2650R on vermiculite without the need of expensive fermentors offers a reliable process for manufacturing bacterial inoculants in developing countries. Under field conditions, the horizontal and vertical movement of NBRI2650R was restricted to 30 and 60 cm, respectively, and the strain could not survive in the field during the 7 months before the chickpea could be planted for next cropping season. Field trials conducted at Jhansi, Kanpur, and Pantnagar resulted in higher grain yield increase in the bacteria-treated seed compared with the nonbacterized control. Seed and furrow treatment of the two chickpeas ('Radhey' and 'H-208') at Pantnagar resulted in significantly (P = 0.05) greater seedling mortality in nonbacterized seedlings compared with bacterized ones. The seed dry weight and yield for each variety were also significantly higher in bacterized seedlings than in nonbacterized ones. The population of NBRI2650R persisted throughout the growing season of chickpea in the range of 5.4-6.4 log10 CFU/g root.

Drug Resistance, Bacterial↗
PubMed1996

Biomass relationship to growth and phosphate uptake of Pseudomonas fluorescens, Escherichia coli and Acinetobacter radioresistens in mixed liquor medium.

The ability of Pseudomonas fluorescens, Escherichia coli and Acinetobacter radioresistens to remove phosphate during growth was related to the initial biomass as well as to growth stages and bacterial species. Phosphate was removed by these bacteria under favourable conditions as well as under unfavourable conditions of growth. Experiments showed a relationship between a high initial cell density and phosphate uptake. More phosphate was released than removed when low initial cell densities (10(2)-10(5) cells ml-1) were used. At a high initial biomass concentration (10(8) cells ml-1), phosphate was removed during the lag phase and during logarithmic growth by P. fluorescens. Escherichia coli, at high initial biomass concentrations (10(7) cells ml-1), accumulated most of the phosphate during the first hour of the lag phase and/or during logarithmic growth and in some cases removed a small quantity of phosphate during the stationary growth phase. Acinetobacter radioresistens, at high initial cell densities (10(6), 10(7) cells ml-1) removed most of phosphate during the first hour of the lag phase and some phosphate during the stationary growth phase. Pseudomonas fluorescens removed phosphate more than A. radioresistens and E. coli with specific average ranges from 3.00-28.50 mg L-1 compared to average ranges of 4.92-17.14 mg L-1 for A. radioresistens and to average ranges of 0.50-8.50 mg L-1 for E. coli.

Acinetobacter↗
PubMed2003

Degradation of 3,4-dichloro- and 3,4-difluoroaniline by Pseudomonas fluorescens 26-K.

3,4-Dichloro- and 3,4-difluoroanilines were degraded by Pseudomonas fluorescens 26-K under aerobic conditions. In the presence of glucose strain degraded 170 mg/L of 3,4-dichloroaniline (3,4-DCA) during 2-3 days. Increasing of toxicant concentration up to 250 mg/L led to degradation of 3,4-DCA during 4 days and its intermediates during 5-7 days. Without cosubstrate and nitrogen source degradation of 3,4-DCA took place too, but more slowly--about 40% of toxicant at initial concentration 75 mg/L was degraded during 15 days. 3,4-Difluoroaniline (3,4-DFA) (initial concentration 170 mg/L) was degraded by Pseudomonas fluorescens 26-K during 5-7 days. The strain was able to completely degrade up to 90 mg/L of 3,4-DFA, without addition of cosubstrate and nitrogen during 15 days. Degradation of fluorinated aniline was accompanied by intensive defluorination. Activity of catechol 2,3-dioxygenase (C2,3DO) (0.230 micromol/min/mg of protein) was found in the culture liquid of the strain, grown with 3,4-DCA and glucose. This fact, as well as, the presence of 3-chloro-4-hydroxyaniline as a metabolite suggested that 3,4-DCA degradation pathway includes dehalogenation and hydroxylation of aromatic ring followed by its subsequent cleaving by C2,3DO. On the contrary, activity of catechol 1,2-dioxygenase (C1,2DO) (0.08 micromol/min/mg of protein) was found in the cell-free extract of biomass grown on 3,4-DFA. 3-Fluoro-4-hydroxyaniline as intermediate was found in this cell-free extract.

Aniline Compounds↗
PubMedDate not supplied

[Regulation of the adhesion of Pseudomonas fluorescens cells to glass by culture-produced volatile compounds].

The effect of the gaseous metabolites of one Pseudomonas fluorescens culture on the attachment of cells of another Pseudomonas fluorescens culture to glass was studied. Gaseous metabolites increased the number of unattached cells by 10-30% and the mean residence time of cells attached to glass by 100%. These effects were presumably due to the yet unidentified compound, which we called volatile antiadhesin. This compound could be adsorbed by activated carbon and HAYESEP-Q adsorbent.

Air↗
PubMed1998

Naphthalene uptake by a Pseudomonas fluorescens isolate.

The uptake of naphthalene has been investigated in the metabolizing cells of Pseudomonas fluorescens utilizing [1-14C]naphthalene. The uptake displayed an affinity constant (Kt) of 11 microM and a maximal velocity (Vmax) of 17 nmol.h-1.mg-1 cellular dry weight. Naphthalene uptake was not observed in a mutant strain, TG-5, which was unable to utilize naphthalene as a sole source of carbon for growth. Uptake was significantly inhibited (approximately 90%) by the presence of growth-inhibiting levels of either azide or 2,4-dinitrophenol and was sensitive to the presence of structural analogues of naphthalene. The intracellular levels of ATP were not significantly reduced by the presence of either azide or 2,4-dinitrophenol. The presence of alpha-naphthol was found to noncompetitively inhibit naphthalene uptake, displaying a Ki of 0.041 microM. It is concluded that the first step in the utilization of naphthalene by Pseudomonas fluorescens is its transport into the cell by a specific energy-linked transport system.

2,4-Dinitrophenol↗
PubMed1993

Some factors affecting airborne survival of Pseudomonas fluorescens indoors.

The effect of relative humidity (RH) on the airborne survival of a Pseudomonas fluorescens strain was studied at 20, 40, 60 and 80% RH indoors. The aero-stable spore of Bacillus subtilis subsp. niger, used as a tracer of physical losses was compared with a light scattering particle counter, as there were doubts about the reliability of the spore as tracer. The Rion counter was validated before use and found to give a good estimate of relative physical losses providing spray suspensions contained between 10(7) and 10(9) cfu ml-1 cells and that the humidity was not more than 80% RH. Pseudomonas fluorescens, strain P+S+, suspended in distilled water survived best at mid humidities and least at 80% RH. When suspended in 1% glycerol there was an apparent 'increase' in viability after an initial rapid reduction. This was thought to be due to delay in equilibration of glycerol in the cell membrane after concentration on dehydration. The cells were thought to be unstable and sensitive to the stress of rehydration before equilibration occurred. The findings are discussed in relation to Cox's theories of outer membrane damage on aerosolization.

Aerosols↗
PubMed2005

The nadA gene of Pseudomonas fluorescens PGPR strain 267.1.

An insertion mutant of Pseudomonas fluorescens PGPR strain 267.1 was found to be auxotrophic for niacin (nicotinic acid) and could not synthesize quinolinic acid. The Tn5 interrupted gene was cloned and sequenced. The cloned fragment contained an open reading frame, nadA, capable of encoding a 359-amino-acid protein (39.0 kDa) with substantial identity to various bacterial quinolinate synthetases. The nadA gene complemented quinolinic acid synthesis deficiency and niacin auxotrophy of the P. fluorescens 106 P nadA mutant.

Genes, Bacterial↗
PubMed1996

Optimization studies of components in enzymatic cholesterol reagents containing cholesterol oxidase from Nocardia erythropolis, Streptomyces sp, or Pseudomonas fluorescens.

Although enzymatic methods for serum cholesterol determination are widely used in clinical laboratories, little is known about the optimization of each component in enzymatic reagents. We investigated the optimal components in the reagents containing cholesterol oxidase isolated from Nocardia erythropolis, Streptomyces sp, or Pseudomonas fluorescens. The optimal components in the reagents are: cholesterol oxidase 250 (Nocardia erythropolis), 250 (Streptomyces sp), or 300 (Pseudomonas fluorescens) U/L, cholesterol esterase 200 U/L, peroxidase 10,000 U/L, sodium cholate 3 mmol/L, 4-aminoantipyrine 0.5 mmol/L, phenol 20 mmol/L, Triton X-100 2 mL/L, and phosphate buffer, pH 7.0. Lower reaction sensitivity and lower cholesterol linearity, < 18.1 mmol/L (700 mg/dL), could be obtained by using lower components than those suggested above. Pseudomonas fluorescens were an improper source for cholesterol oxidase; either Nocardia erythropolis or Streptomyces was suitable cholesterol oxidase. We prefer using Streptomyces sp cholesterol oxidase because of its economical cost and longest reagent stability. Sodium cholate must be included in the enzymatic reagent to prevent turbidity. However, sodium cholate of > 5 mmol/ L will suppress the reaction resulting in low cholesterol linearity.

Ampyrone↗
PubMed1994

A spontaneous point mutation in the aac(6')-Ib' gene results in altered substrate specificity of aminoglycoside 6'-N-acetyltransferase of a Pseudomonas fluorescens strain.

The aac(6')-Ib' gene from Pseudomonas fluorescens BM2687, encoding an aminoglycoside 6'-N-acetyltransferase type II which confers resistance to gentamicin but not to amikacin, was characterized. Nucleotide sequence determination indicated total identity between aac(6')-Ib' and the aac(6')-Ib gene from Pseudomonas aeruginosa BM2656 [1] with the exception of a C-to-T transition that results in a serine to leucine substitution at position 83 of the deduced polypeptide. The aac(6')-Ib gene specifies a type I enzyme which confers resistance to amikacin but not to gentamicin [2]. It thus appears that the point mutation detected is responsible for enzymic altered substrate specificity.

Acetyltransferases↗
PubMed1994

Transposon Tn5 mutagenesis of pseudomonas fluorescens to isolate mutants deficient in antibacterial activity.

Pseudomonas fluorescens was subjected to insertion mutagenesis studies using the transposon Tn5-GM to generate mutants deficient in antibacterial activity minus mutants. The transposon located on the temperature-sensitive plasmid pCHR84 was conjugally transferred into the non-pathogenic pseudomonad using the triparental mating procedure. Random integration of Tn5-GM into the chromosome of P. fluorescens was achieved by heat treatment of the transformed cells at 42 degrees C. Approximately 2% of transconjugants revealed an auxotrophic phenotype indicating efficient integration of the employed transposon into the chromosome of P. fluorescens. One transposon insertion mutant was obtained showing an antibacterial activity minus phenotype. This mutant (MM-7) was found to be defective in the production of an unidentified antibacterial compound against B. subtilis. These results introduce Tn5 transposon mutagenesis as a new useful tool for the molecular analysis of P. fluorescens.

Antibiosis↗
PubMed1970

Isolation and properties of a beta-alanine transaminaseless mutant of Pseudomonas fluorescens.

beta-Alanine catabolism in Pseudomonas fluorescens is initiated by the enzyme beta-alanine transaminase. We have isolated mutants which fail to produce this enzyme and thus cannot grow on beta-alanine as the sole nitrogen source. The accumulation of beta-alanine-1-(14)C has been studied in one of these mutants, strain 67, and in the wild type. In the mutant, beta-alanine remains in a stable intracellular pool, whereas in the wild type conversion of beta-alanine to an intermediate, presumably malonate semialdehyde, and to CO(2) can be detected. The membrane transport system for beta-alanine can be conveniently studied in this transaminaseless mutant.

Alanine↗
PubMed2002

Induction of phenylpropanoid metabolism by Pseudomonas fluorescens against tomato spotted wilt virus in tomato.

Pseudomonas fluorescens (two native strains, one collection strain and their strain mixtures in all possible combinations) when applied through seed, seedling dip, soil and on leaf significantly reduced the tomato spotted wilt virus (TSWV) disease. In P. fluorescens-treated plants, the peroxidase and phenylalanine ammonia-lyase activity increased. Accumulation of phenolic compounds and lignin were shown to be increased in the P. fluorescens-treated plants. Isoperoxidase native PAGE indicated that the peroxidase isoforms in tomato plants induced by fluorescent pseudomonads were different from the control plants; this suggests that the general phenylpropanoid pathway is probably stimulated in tomato plants treated which in turn led to significant reduction in TSWV.

Electrophoresis, Polyacrylamide Gel↗
PubMed1993

Cloning, nucleotide sequence, and expression of a p-hydroxybenzoate hydroxylase isozyme gene from Pseudomonas fluorescens.

A gene encoding for a putative isozyme of p-hydroxy-benzoate hydroxylase (PHBH) has been isolated from Pseudomonas fluorescens (ATCC 13525). A comparison of the translated amino acid sequence with that of the known PHBH from P. fluorescens revealed that the new enzyme contains 3 additional amino acids and has 73% absolute homology to the previously known enzyme; conservation of secondary and active-site structures implied that the isozyme and known enzyme share the same general tertiary structure. Subsequent expression of the isozyme in Escherichia coli produced an enzyme with a specific activity about half that of the previously characterized PHBHs from P. fluorescens and Pseudomonas aeruginosa; in addition, somewhat weaker binding affinities for both NADPH and p-hydroxybenzoate were observed. Speculations are made on the reason for the existence of the isozyme, which does not appear to be expressed routinely in P. fluorescens.

Amino Acid Sequence↗
PubMed1995

Specificity of an esterase (XYLD) from Pseudomonas fluorescens subsp. cellulosa.

Activity of an esterase from Pseudomonas fluorescens subsp. cellulosa (XYLD) on an insoluble feruloylated hemicellulose substrate (de-starched wheat bran) was dependent on the source of added endo-xylanase. The esterase exhibited high selectivity for the nature, position of linkage and size of the feruloylated oligosaccharides generated by hydrolysis of the hemicellulose. Increased affinity of XYLD with increasing size of the oligosaccharide substrate suggests that optimal activity is observed on substrates with at least 4 sugars.

Coumaric Acids↗
PubMed1987

[3-(3-hydroxy-2,3-dimethyl-5-oxoprolyl)amino-3,6-dideoxy-D-glucose: a new amino sugar of the antigenic polysaccharide from Pseudomonas fluorescens].

O-specific polysaccharide chain of the Pseudomonas fluorescens lipopolysaccharide is composed of 6-deoxy-L-talose, N-acetyl-D-fucosamine and N-acyl-3,6-dideoxy-D-glucose. Analysis of the latter sugar, obtained from the polysaccharide hydrolysate, by 1H NMR (including NOE), 13C NMR, and FAB mass spectrometry proved the unusual N-acyl substituent to be a 3-hydroxy-2,3-dimethyl-5-oxoproline residue.

Amino Sugars↗
PubMed2004

Phylogenetic relationships between environmental and clinical isolates of Pseudomonas fluorescens and related species deduced from 16S rRNA gene and OprF protein sequences.

The major surface protein of the genus Pseudomonas, OprF, is a non-specific porin that plays an important role in maintenance of cell shape, in growth in a low osmolarity environment, and in adhesion to various supports. The objectives of our study were (i) to carry out a comparative analysis of phylogenies obtained from the OprF protein and from the 16S rRNA gene in 41 isolates from various sources (water, soil, milk and the hospital) and (ii) to investigate the physiological characteristics correlated with the phylogeny of OprF. We report here an important incongruence between the phylogenies of the 16S rRNA gene and the OprF protein. Phylogenetic analysis of 16S rRNA genes grouped Pseudomonas fluorescens isolates into one cluster (termed fluorescens r-cluster) whilst the phylogeny of the OprF protein divided Pseudomonas fluorescens isolates into two quite distinct clusters (termed fluorescens 1 o-cluster and fluorescens 2 o-cluster) that may be related to the original habitat of the strain. The fluorescens 1 o-cluster contained the majority of non-rhizospheric soil isolates, while the fluorescens 2 o-cluster contained all our clinical isolates and most of the rhizospheric isolates (which are fixed to the roots). In order to check this correlation, we studied two physiological characteristics: the range of growth temperature and the capacity for non-specific adhesion to polystyrene. The temperature range study for strains did not explain the existence of the two o-clusters but it did confirm the capacity of certain P. fluorescens strains to grow at 37 degrees C. The adhesion capacities of the isolates in the two o-clusters seems to be correlated with ecological niche.

Bacterial Adhesion↗