Ornicorrugatin, a new siderophore from Pseudomonas fluorescens AF76

From a pyoverdin-negative mutant of Pseudomonas fluorescens AF76 a new lipopeptidic siderophore (ornicorrugatin) could be isolated. It is structurally related to the siderophore of Pseudomonas corrugata differing in the replacement of one Dab unit by Orn.     

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.     

Dialysis fermentation. I. Enhanced production of salicylic acid from naphthalene by Pseudomonas fluorescens

The conversion of naphthalene to salicylic acid by Pseudomonas fluorescens was studied as a model for dialysis fermentation. In a demonstration experiment, the continual removal of the product by dialysis and by intermittent replenishment of the dialysate reservoir caused cyclical changes in the concentrations of viable cells and product. The cumulative total amount of both cell mass and salicylate, however, continued to increase steadily until the experiment was terminated after 15 days. At this time the rate of product formation was highest and still increasing, although less than 10% of the cells were viable. The terminal amount of salicylate was about 20-fold greater than the maximum reached in the control fermentation, and was calculated to be 2.6-fold more productive even if the control were optimally recycled. Methods were projected to achieve still further improvements.     

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.     

Pseudomonas fluorescens CHA0 produces enantio-pyochelin, the optical antipode of the Pseudomonas aeruginosa siderophore pyochelin

The siderophore pyochelin is made by a thiotemplate mechanism from salicylate and two molecules of cysteine. In Pseudomonas aeruginosa, the first cysteine residue is converted to its D-isoform during thiazoline ring formation whereas the second cysteine remains in its L-configuration, thus determining the stereochemistry of the two interconvertible pyochelin diastereoisomers as 4'R, 2'R, 4'R (pyochelin I) and 4'R, 2'S, 4'R (pyochelin II). Pseudomonas fluorescens CHA0 was found to make a different stereoisomeric mixture, which promoted growth under iron limitation in strain CHA0 and induced the expression of its biosynthetic genes, but was not recognized as a siderophore and signaling molecule by P. aeruginosa. Reciprocally, pyochelin promoted growth and induced pyochelin gene expression in P. aeruginosa, but was not functional in P. fluorescens. The structure of the CHA0 siderophore was determined by mass spectrometry, thin-layer chromatography, NMR, polarimetry, and chiral HPLC as enantio-pyochelin, the optical antipode of the P. aeruginosa siderophore pyochelin. Enantio-pyochelin was chemically synthesized and confirmed to be active in CHA0. Its potential biosynthetic pathway in CHA0 is discussed.     

Environmental factors modulating antibiotic and siderophore biosynthesis by Pseudomonas fluorescens biocontrol strains.

Understanding the environmental factors that regulate the biosynthesis of antimicrobial compounds by disease-suppressive strains of Pseudomonas fluorescens is an essential step toward improving the level and reliability of their biocontrol activity. We used liquid culture assays to identify several minerals and carbon sources which had a differential influence on the production of the antibiotics 2,4-diacetylphloroglucinol (PHL), pyoluteorin (PLT), and pyrrolnitrin and the siderophores salicylic acid and pyochelin by the model strain CHA0, which was isolated from a natural disease-suppressive soil in Switzerland. Production of PHL was stimulated by Zn2+, NH4Mo2+, and glucose; the precursor compound mono-acetylphloroglucinol was stimulated by the same factors as PHL. Production of PLT was stimulated by Zn2+, Co2+, and glycerol but was repressed by glucose. Pyrrolnitrin production was increased by fructose, mannitol, and a mixture of Zn2+ and NH4Mo2+. Pyochelin production was increased by Co2+, fructose, mannitol, and glucose. Interestingly, production of its precursor salicylic acid was increased by different factors, i.e., NH4Mo2+, glycerol, and glucose. The mixture of Zn2+ and NH4Mo2+ with fructose, mannitol, or glycerol further enhanced the production of PHL and PLT compared with either the minerals or the carbon sources used alone, but it did not improve siderophore production. Extending fermentation time from 2 to 5 days increased the accumulation of PLT, pyrrolnitrin, and pyochelin but not of PHL. When findings with CHA0 were extended to an ecologically and genetically diverse collection of 41 P. fluorescens biocontrol strains, the effect of certain factors was strain dependent, while others had a general effect. Stimulation of PHL by Zn2+ and glucose was strain dependent, whereas PLT production by all strains that can produce this compound was stimulated by Zn2+ and transiently repressed by glucose. Inorganic phosphate reduced PHL production by CHA0 and seven other strains tested but to various degrees. Production of PLT but not pyrrolnitrin by CHA0 was also reduced by 100 mM phosphate. The use of 1/10-strength nutrient broth-yeast extract, compared with standard nutrient broth-yeast extract, amended with glucose and/or glycerol resulted in dramatically increased accumulations of PHL (but not PLT), pyochelin, and salicylic acid, indicating that the ratio of carbon source to nutrient concentration played a key role in the metabolic flow. The results of this study (i) provide insight into the biosynthetic regulation of antimicrobial compounds, (ii) limit the number of factors for intensive study in situ, and (iii) indicate factors that can be manipulated to improve bacterial inoculants.     

Role of salicylic acid in systemic resistance induced by Pseudomonas fluorescens against Fusarium oxysporum f. sp. ciceri in chickpea

Selected isolates of Pseudomonas fluorescens (Pf1-94, Pf4-92, Pf12-94, Pf151-94 and Pf179-94) and chemical resistance inducers (salicylic acid, acetylsalicylic acid, DL-norvaline, indole-3-carbinol and lichenan) were examined for growth promotion and induced systemic resistance against Fusarium wilt of chickpea. A marked increase in shoot and root length was observed in P. fluorescens treated plants. The isolates of P. fluorescens systemically induced resistance against Fusarium wilt of chickpea caused by Fusarium. oxysporum f.sp. ciceri (FocRs1), and significantly (P = 0.05) reduced the wilt disease by 26-50% as compared to control. Varied degree of protection against Fusarium wilt was recorded with chemical inducers. The reduction in disease was more pronounced when chemical inducers were applied with P. fluorescens. Among chemical inducers, SA showed the highest protection of chickpea seedlings against wilting. Fifty two- to 64% reduction of wilting was observed in soil treated with isolate Pf4-92 along with chemical inducers. A significant (P = 0.05; r = -0.946) negative correlation was observed in concentration of salicylic acid and mycelial growth of FocRs1 and at a concentration of 2000 microg ml(-1) mycelial growth was completely arrested. Exogenously supplied SA also stimulated systemic resistance against wilt and reduced the disease severity by 23% and 43% in the plants treated with 40 and 80 microg ml(-1) of SA through root application. All the isolates of P. fluorescens produced SA in synthetic medium and in root tissues. HPLC analysis indicated that Pf4-92 produced comparatively more SA than the other isolates. 1700 to 2000 nanog SA g(-1) fresh root was detected from the application site of root after one day of bacterization whereas, the amount of SA at distant site ranged between 400-500 nanog. After three days of bacterization the SA level decreased and was found more or less equal at both the detection sites.     

The metabolism of aluminum citrate and biosynthesis of oxalic acid in Pseudomonas fluorescens

(13)CNMR and (1)HNMR studies revealed that aluminum citrate (Al-citrate) was metabolized intracellularly and that oxalic acid was an important product in the Al-stressed cells. This dicarboxylic acid was produced via the oxidation of glyoxylate, a precursor generated through the cleavage of isocitrate. In the control cells, citrate was biotransformed essentially with the aid of regular tricarboxylic cycle (TCA) enzymes. However, these control cells were able neither to uptake nor to metabolize Al-citrate. Al-stressed cells obtained at 38-40 h of growth showed maximal Al-citrate uptake and biotransforming activities. At least a fourfold increase in the activity of the enzyme isocitrate lyase (ICL, E. C. 4.1.3.1) has been observed in the Al-stressed cells compared with the control cells. The transport of Al-citrate was sensitive to p-dinitrophenol and sodium azide, but not to dicyclohexylcarbodiimide. Experiments with the dye 9-aminoacridine revealed that the translocation of Al-citrate led to an increase in intracellular pH. Thus, it appears that after the uptake of Al-citrate, this complex is metabolized intracellularly.     

Deleterious impact of a virulent bacteriophage on survival and biocontrol activity of Pseudomonas fluorescens strain CHAO in natural soil

Many biotic and abiotic factors affect the persistence and activity of beneficial pseudomonads introduced into soil to suppress plant diseases. One such factor may be the presence of virulent bacteriophages that decimate the population of the introduced bacteria, thereby reducing their beneficial effect. We have isolated a lytic bacteriophage (phi)GP100) that specifically infects the biocontrol bacterium Pseudomonas fluorescens CHA0 and some closely related Pseudomonas strains. phiGP100 was found to be a double-stranded-DNA phage with an icosahedral head, a stubby tail, and a genome size of approximately 50 kb. Replication of phiGP100 was negatively affected at temperatures higher than 25 degrees C. phiGP100 had a negative impact on the population size and the biocontrol activity of P. fluorescens strain CHA0-Rif (a rifampicin-resistant variant of CHA0) in natural soil microcosms. In the presence of phiGP100, the population size of strain CHA0-Rif in soil and on cucumber roots was reduced more than 100-fold. As a consequence, the bacterium's capacity to protect cucumber against a root disease caused by the pathogenic oomycete Pythium ultimum was entirely abolished. In contrast, the phage affected neither root colonization and nor the disease suppressive effect of a phiDGP100-resistant variant of strain CHA0-Rif. To our knowledge, this study is the first to illustrate the potential of phages to impair biocontrol performance of beneficial bacteria released into the natural soil environment.     

Neutral amino acid transport in Pseudomonas fluorescens

Membrane transport of beta-alanine, l-alanine, and l-proline was studied in a beta-alanine transaminaseless mutant (strain 67) of Pseudomonas fluorescens. In this mutant beta-alanine is metabolically inert, and it was therefore possible to demonstrate active transport of this substrate in the absence of intracellular catabolism. The permease which catalyzes the uptake of beta-alanine also transports l-proline and l-alanine. This common transport system was distinguished from permeases which transport only l-alanine and only l-proline by competition studies in strain 67 and by studies of transport specificity in a permeaseless mutant (strain 67/4MTR).     

Quinolobactin, a new siderophore of Pseudomonas fluorescens ATCC 17400, the production of which is repressed by the cognate pyoverdine

Transposon mutant strain 3G6 of Pseudomonas fluorescens ATCC 17400 which was deficient in pyoverdine production, was found to produce another iron-chelating molecule; this molecule was identified as 8-hydroxy-4-methoxy-quinaldic acid (designated quinolobactin). The pyoverdine-deficient mutant produced a supplementary 75-kDa iron-repressed outer membrane protein (IROMP) in addition to the 85-kDa IROMP present in the wild type. The mutant was also characterized by substantially increased uptake of (59)Fe-quinolobactin. The 75-kDa IROMP was produced by the wild type after induction by quinolobactin-containing culture supernatants obtained from the pyoverdine-negative mutant or by purified quinolobactin. Conversely, adding purified wild-type pyoverdine to the growth medium resulted in suppression of the 75-kDa IROMP in the pyoverdine-deficient mutant; however, suppression was not observed when Pseudomonas aeruginosa PAO1 pyoverdine, a siderophore utilized by strain 3G6, was added to the culture. Therefore, we assume that the quinolobactin receptor is the 75-kDa IROMP and that the quinolobactin-mediated iron uptake system is repressed by the cognate pyoverdine.     

Phthalate metabolism in Pseudomonas fluorescens PHK: purification and properties of 4,5-dihydroxyphthalate decarboxylase.

Pseudomonas fluorescens PHK uses 4,5-dihydroxyphthalate as the sole carbon source for o-phthalate catabolism. This intermediate is the substrate for a decarboxylase of the pathway yielding protocatechuate. The decarboxylase was purified to homogeneity by an affinity chromatography procedure in which the reaction product, protocatechuate, was used as a ligand. We describe some properties of the enzyme, including its apparent molecular weight of 420,000 as determined by gel filtration and of 66,000 after sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis, consistent with a hexameric functional protein. The apparent Km for the substrate 4,5-dihydroxyphthalate was 10.4 microM. The characteristics of this enzyme are compared with those described for the isofunctional enzyme from P. testosteroni.     

The metabolism of halogen-substituted benzoic acids by Pseudomonas fluorescens

1. Washed suspensions of Pseudomonas fluorescens, grown with benzoate as sole carbon source, oxidize monohalogenobenzoates in the following descending order of effectiveness: benzoate, fluorobenzoates, chlorobenzoates, bromobenzoates, iodobenzoates. 2. Cells grown on asparagine oxidize benzoate after an adaptive period of 90–120min. This adaptive period is increased by halogenobenzoates in the following approximate descending order of effectiveness: chlorobenzoates, fluorobenzoates (=bromobenzoates), iodobenzoates. This inhibition of adaptation by halogeno analogues depends on the concentration of benzoate and is thus apparently competitive. 3. Cells do not adapt to oxidize the halobenzoates when the halogeno analogues are inducers. However, the fluorobenzoates reduce the lag period taken to form the benzoate-oxidizing system. 4. The halogenobenzoates inhibit adaptation to citrate and nicotinate but not so effectively as benzoate itself. This is presumably a `diauxic' effect. The analogues do not inhibit adaptation to catechol. 5. The halogenobenzoates are not used as sole carbon source for growth nor do they increase growth when cells grow with asparagine as the main carbon source. 6. It is suggested that this inability to use the analogues for growth is due partly to inability of the cells to liberate the halogen and to carry the oxidation to a stage at which carbon may be assimilated and partly to the inhibition of the induction of the oxidizing enzymes.     

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.