The Fe2+ Chelator Proferrorosamine A Is Essential for the Siderophore-Mediated Uptake of Iron by Pseudomonas roseus fluorescens

Pseudomonas roseus fluorescens produces, besides the Fe²⁺ chelator proferrorosamine A, Fe³⁺ -chelating compounds, called siderophores. The production of proferrorosamine A and siderophores by P. roseus fluorescens appears to be controlled in a similar way by the concentration of available iron and by the concentration of dissolved oxygen. The higher the concentration of iron available for the microorganism, the lower the production of both chelating compounds. However, the production of siderophores was much more sensitive to iron availability than was proferrorosamine A production. Proferrorosamine A and siderophores were only produced in minimal medium C if the concentration of dissolved oxygen ranged from 4.5 to 2.0 ppm. At higher or lower concentrations, none of the iron-chelating compounds were produced. Furthermore, it has been shown that proferrorosamine-negative Tn5 mutants of P. roseus fluorescens were able to form siderophores only under iron-limiting conditions when proferrorosamine A was added to the medium. Our data suggest that proferrorosamine A production is essential for siderophore synthesis by P. roseus fluorescens; the production of siderophores occurred only when proferrorosamine A was present.     

Biosynthesis and cytoplasmic accumulation of a chlorinated catechol pigment during 3-chlorobenzoate aerobic co-metabolism in Pseudomonas fluorescens

A strain of Pseudomonas fluorescens was capable of co-metabolizing 3-chlorobenzoic acid with the production of a chlorinated catechol black pigment. A peroxidase and another enzymatic activity referred to as a polyphenol oxidase were found to be involved in the oxidation of 4-chlorocatechol to 4-chloro-1,2-benzoquinone, i.e. in the production of highly reactive substrates for pigment formation. Therefore, P. fluorescens cells were seen to take an active part not only in 3-chlorobenzoate mineralization but also in overall pigment production. pH was found to be a key parameter in the regulation of the activity of P. fluorescens oxidoreductive enzymes. Ultrastructural investigations showed that electron dense granules of pigment were distributed throughout the cytoplasm of Pseudomonas fluorescens cells grown in presence of 3-chlorobenzoate, as confirmed also by Thiéry cytochemical investigations.In these cells, an extensive contraction of the cytoplasm as well as a significant damage to the cell wall after two days of incubation, suggested that pigment production caused a premature death of the cells accompanied by the leakage of the cell content. Pigment production seemed to occur mostly in the cytoplasmic context where the electron dense material accumulates until it is released in the medium after the cell lysis.Abbreviations 3-CBA 3-chlorobenzoic acid - BA benzoic acid - 4-CC 4-chlorocatechol - 3-CC 3-chlorocatechol - MBTH 3-methyl-2-benzothiazolinone hydrazone - l-DOPA l-3,4-dihydroxyphenyl-alanine - SPB sodium phosphate buffer     

A simple assay for fluorescent siderophores produced by Pseudomonas species and an efficient isolation of pseudobactin

Several iron binding metabolites (siderophores) of Pseudomonas fluorescens B10 (JL-3133) have been detected using C₁₈ reverse phase HPLC coupled with photodiode array detection methods. This analysis utilized a volatile mobile phase of 90% 20 mm NH₄HCO₃/10% MeOH, pH 6.5. It has been shown to be applicable to other P. fluorescens strains for the detection of related metabolites. Direct scale-up of the analytical HPLC conditions allowed for the efficient preparative isolation of pseudobactin, the principle siderophore produced by P. fluorescens B10 (JL-3133).     

Interactions between rhizosphere microorganisms under iron limitation

Bacillus polymyxa, Pseudomonas cepacia and Pseudomonas fluorescens are present in the rhizosphere of many crop plants. Little is known about microbial interactions in the rhizosphere. We investigated the type of interaction between these species under iron limitation. We found that, in mixed batch cultures, P. cepacia stimulates the growth of B. polymyxa and this stimulation can be observed also in low iron medium. Cell-free supernatants of cultures of P. fluorescens with various amounts of the siderophore pyoverdine also stimulate the growth of B. polymyxa. In this case we observed a positive correlation between pyoverdine concentration and growth stimulation. Purified pyoverdine also affects positively the growth of B. polymyxa.     

Coimmobilized whole cells of Pseudomonas reptilivora and Micrococcus glutamicus in calcium alginate gel for the production of L-glutamic acid

A novel method of coimmobilized whole cells of Pseudomonas reptilivora and Micrococcus glutamicus, entrapped in calcium alginate beads have been used for the production of L-glutamic acid in a single stage fermentation process, using selected production medium enriched with glucose as substrate. The results obtained were compared with the L-glutamic acid production by free cells of Micrococcus glutamicus and by mixed culture of Pseudomonas reptilivora and Micrococcus glutamicus. The yield of glutamic acid obtained with mixed culture is relatively more than that the yield obtained with Micrococcus glutamicus alone. The properties of coimmobilized whole cells of Pseudomonas reptilivora and Micrococcus glutamicus in calcium alginate gel matrix have been investigated thoroughly and compared with those of free cells under most suitable conditions of fermentation.     

Detection of siderophores in growing cultures ofPseudomonas spp.

Summary The siderophores produced byPseudomonas fluorescens andP. chlororaphis were detected from the culture supernatants in MM9 and modified King's medium by the universal CAS assay at wavelengths 620–690 nm. The CAS assay was applied to detectPseudomonas siderophores directly in situ, during their production phase, in modified King's medium. Optimum results were detected with a final CAS concentration of 0.025 mM and an iron concentration of 1.25 M. The problems of the method are discussed with respect to the absorbance spectrum, the toxicity of the HDTMA detergent, the influence of the iron concentration and the complexity of media for siderophore production.     

Iron uptake and metabolism in pseudomonads

Pseudomonads are ubiquitous Gram-negative γ proteobacteria known for their extreme versatility and adaptability. Some are plant pathogens (Pseudomonas syringae) which have to survive on the surface of leaves while others can colonize the rhizosphere or survive in soil (Pseudomonas fluorescens, Pseudomonas putida), and one species, Pseudomonas entomophila, is an insect pathogen. The most investigated species, Pseudomonas aeruginosa, is known to be an opportunistic pathogen able to infect plants, nematodes, insects, and mammals, including humans. Like for other bacteria, iron is a key nutrient for pseudomonads. The fluorescent pseudomonads produce siderophores, the best known being the fluorescent high-affinity peptidic pyoverdines. Often diverse secondary siderophores of lower affinity are produced as well (pyochelin, pseudomonin, corrugatins and ornicorrugatins, yersiniabactin, and thioquinolobactin). Reflecting their large capacity of adaptation to changing environment and niche colonization, pseudomonads are able to obtain their iron from heme or from siderophores produced by other microorganisms (xenosiderophores) via the expression of outer membrane TonB-dependent receptors. As expected, iron uptake is exquisitely and hierarchically regulated in these bacteria. In this short review, the diversity of siderophores produced, receptors, and finally the way iron homeostasis is regulated in P. aeruginosa, P. syringae, P. putida, and P. fluorescens, will be presented and, when possible, put in relation with the lifestyle and the ecological niche.     

Modeling of growth kinetics for Pseudomonas spp. during benzene degradation

A modeling study was conducted on growth kinetics of three different strains of Pseudomonas spp. (Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas putida) during benzene degradation to determine optimum substrate concentrations for most efficient biodegradation. Batch tests were performed for eight different initial substrate concentrations to observe cell growth and associated substrate degradation using benzene-adapted cells. Kinetic parameters of both inhibitory (Haldane–Andrews, Aiba–Edwards) and noninhibitory (Monod) models were fitted to the relationship between specific growth rate and substrate concentration obtained from the growth curves. Results showed that half-saturation constant of P. fluorescens was the highest among the three strains, indicating that this strain could grow well at high concentration, while P. putida could grow best at low concentration. The inhibition constant of P. aeruginosa was the highest, implying that it could tolerate high benzene concentration and therefore could grow at a wider concentration range. Estimated specific growth rate of P. putida was lower, but half-saturation constant was higher than those from literature study due to high substrate concentration range used in this study. These two kinetic parameters resulted in substantial difference between Monod- and Haldane-type models, indicating that distinction should be made in applying those models.     

Effect of iron on the production of pyoverdine and outer membrane proteins ofPseudomonas aeruginosa PA01

Pseudomonas aeruginosa PA01 grown under iron-deficient conditions excreted a yellowish-green pigment, pyoverdine, which has been reported as a siderophore of this bacterium. Addition of iron to the culture medium increased bacterial growth but repressed the production of pyoverdine. Iron levels of the culture medium also had a significant role in the composition of outer membrane proteins ofP. aeruginosa PA01.     

Siderophore-mediated iron acquisition in the entomopathogenic bacterium Pseudomonas entomophila L48 and its close relative Pseudomonas putida KT2440

Pseudomonas entomophila L48 is a recently identified entomopathogenic bacterium which, upon ingestion, kills Drosophila melanogaster, and is closely related to P. putida. The complete genome of this species has been sequenced and therefore a genomic, genetic and structural analysis of the siderophore-mediated iron acquisition was undertaken. P. entomophila produces two siderophores, a structurally new and unique pyoverdine and the secondary siderophore pseudomonine, already described in P. fluorescens species. Structural analysis of the pyoverdine produced by the closely related P. putida KT2440 showed that this strain produces an already characterised pyoverdine, but different from P. entomophila, and no evidence was found for the production of a second siderophore. Growth stimulation assays with heterologous pyoverdines demonstrated that P. entomophila is able to utilize a large variety of structurally distinct pyoverdines produced by other Pseudomonas species. In contrast, P. putida KT2440 is able to utilize only its own pyoverdine and the pyoverdine produced by P. syringae LMG 1247. Our data suggest that although closely related, P. entomophila is a more efficient competitor for iron than P. putida.     

Colonization behaviour of Pseudomonas fluorescens and Sinorhizobium meliloti in the alfalfa (Medicago sativa) rhizosphere

The colonization ability of Pseudomonas fluorescens F113rif in alfalfa rhizosphere and its interactions with the alfalfa microsymbiont Sinorhizobium meliloti EFB1 has been analyzed. Both strains efficiently colonize the alfalfa rhizosphere in gnotobiotic systems and soil microcosms. Colonization dynamics of F113rif on alfalfa were similar to other plant systems previously studied but it is displaced by S. meliloti EFB1, lowering its population by one order of magnitude in co-inoculation experiments. GFP tagged strains used to study the colonization patterns by both strains indicated that P. fluorescens F113rif did not colonize root hairs while S. meliloti EFB1 extensively colonized this niche. Inoculation of F113rif had a deleterious effect on plants grown in gnotobiotic systems, possibly because of the production of HCN and the high populations reached in these systems. This effect was reversed by co-inoculation. Pseudomonas fluorescens F113 derivatives with biocontrol and bioremediation abilities have been developed in recent years. The results obtained support the possibility of using this bacterium in conjunction with alfalfa for biocontrol or rhizoremediation technologies.     

An integrated approach for the evaluation of biological control of the complex Polymyxa betae/Beet Necrotic Yellow Vein Virus,by means of seed inoculants

Rhizomania is an extremely severe sugarbeet disease caused by the complex Polymyxa betae/Beet Necrotic Yellow Vein Virus (BNYVV). A relatively small number of recently introduced sugarbeet cultivars characterized by a high tolerance to rhizomania are available on the market. An integrated approach was therefore developed using Pseudomonas fluorescens biological control agents (BCAs) in order to improve yield performance of cultivars characterized by a medium tolerance to the disease. A genetically modified biological control agent, Pseudomonas fluorescens F113Rif (pCUGP), was developed for enhanced production of the antimicrobial metabolite 2,4-diacetylphloroglucinol (Phl) and lacking an antibiotic resistance marker gene, making the strain suitable for field release. The ability of synthetic Phl and P. fluorescens F113Rif (pCUGP) to antagonize the fungal vector, P. betae, was assessed in microcosm trials. Results encouraged the preparation of multiple field trials in a soil naturally infested with P. betae/BNYVV, to determine the biocontrol efficacy of P. fluorescens F113Rif (pCUGP) and to assess its impact on sugarbeet yield and quality and on the indigenous microbial population. While the colonization ability of P. fluorescens F113Rif (pCUGP) was satisfactory at sugarbeet emergence (2.5×10⁶ CFU g^–1 root), control of rhizomania was not achieved. Inoculation of sugarbeet with Pseudomonas fluorescens F113Rif (pCUGP) did not affect crop yield and quality nor affect the numbers of selected microbial populations.     

Characterization of monoterpene biotransformation in two pseudomonads

Aims: To study the metabolic profile of Pseudomonas rhodesiae and Pseudomonas fluorescens in water–organic solvent systems using terpene substrates for both growth and biotransformation processes and to determine the aerobic or anaerobic status of these degradation pathways. Materials and Methods: Substrates from pinene (α‐pinene, α‐pinene oxide, β‐pinene, β‐pinene oxide, turpentine) and limonene (limonene, limonene‐1,2‐oxide, orange peel oil) families were tested. For the bioconversion, the terpene‐grown biomass was concentrated and used either as whole cells or as a crude enzymatic extract. Conclusion: Pseudomonas rhodesiae was the most suitable biocatalyst for the production of isonovalal from α‐pinene oxide and did not metabolize limonene. Pseudomonas fluorescens was a more versatile micro‐organism and metabolized limonene in two different ways. The first (anaerobic, cofactor‐independent, noninducible) allowed limonene elimination by synthesizing α‐terpineol. The second (aerobic, cofactor‐dependent) involved limonene‐1,2‐oxide as an intermediate for energy production through a β‐oxidation process. Significance and Impact of the Study: Enzymatic isomerization of β‐ to α‐pinene was described for the first time for both strains. Alpha‐terpineol production by P. fluorescens was very efficient and appeared as a promising alternative for the commercial production of this bioflavour.     

Population dynamics of a dual Pseudomonas putida–Pseudomonas fluorescens biofilm in a capillary bioreactor

Most biofilm studies employ single species, yet in nature biofilms exist as mixed cultures, with inevitable effects on growth and development of each species present. To investigate how related species of bacteria interact in biofilms, two Pseudomonas spp., Pseudomonas fluorescens and Pseudomonas putida, were cultured in capillary bioreactors and their growth measured by confocal microscopy and cell counting. When inoculated in pure culture, both bacteria formed healthy biofilms within 72?h with uniform coverage of the surface. However, when the bioreactors were inoculated with both bacteria simultaneously, P. putida was completely dominant after 48?h. Even when the inoculation by P. putida was delayed for 24?h, P. fluorescens was eliminated from the capillary within 48?h. It is proposed that production of the lipopeptide putisolvin by P. putida is the likely reason for the reduction of P. fluorescens. Putisolvin biosynthesis in the dual-species biofilm was confirmed by mass spectrometry.     

Effect of aeration on ethylene production by soil bacteria and soil samples cultivated in a closed system

Summary Ethylene production was studied in shaken cultures ofPseudomonas putida andPseudomonas fluorescens isolated from soil and in unsterile garden soil samples moistened to 60% of the water holding capacity. The highest ethylene accumulation in bacterial cultures was reached under conditions of delayed aeration,i.e. when the culture was closed and the aeration started after the oxygen content decreased to 4%. The ethylene production rose immediately after the beginning of aeration. Under these conditions ethylene production was inP. fluorescens 2–3 times and in glucosecultivatedP. putida 6 times higher than in the fully aerated cultures. Methionine stimulated ethylene production byP. fluorescens, whereas glucose proved to be more suitable forP. putida. This strain was incapable of growth on methionine as the sole carbon source. Samples of nonsterile garden soil produced the highest amounts of ethylene under anaerobic conditions. Artificial inoculation of soil samples byP. putida resulted in an increase of ethylene formation in samples with delayed aeration. Addition of glucose or glucose with methionine stimulated ethylene production in all soil samples.     

Characterization of a pyoverdine-deficient mutant of Pseudomonas fluorescens impaired in the secretion of extracellular lipase

A mutant of Pseudomonas fluorescens strain B52 deficient in the synthesis of the fluorescent pigment, pyoverdine, was isolated. Absence of pyoverdine and other siderophores was confirmed by gel filtration, a specific siderophore assay, and inhibition studies with the iron chelator EDDA. Both parent and mutant synthesized additional outer membrane proteins in response to iron-limitation. Mutant cells cultured in the absence of iron(III) accumulated ⁵⁵Fe-labeled pyoverdine. The mutant produced extracellular proteinase normally on various media, but was deficient in lipase secretion. Growth of the mutant with partially-purified pyoverdine resulted in a 2.5-fold stimulation of lipase secretion. The mutant grew poorly in deferrated medium; however, the addition of iron(III) stimulated growth. Proteinase secretion in deferrated medium was stimulated over a narrow range of iron(III) concentration, while lipase secretion was only slightly affected. The data suggest that separate regulatory mechanisms exist for the control of proteinase and lipase secretion by iron(III).Contribution No. 768 from the Food Research Centre     

Monitoring Population Size,Activity, and Distribution of gfp-luxAB-Tagged Pseudomonas fluorescens SBW25 during Colonization of Wheat

Increasingly, focus has been directed towards the use of microorganisms as biological control agents to combat fungal disease, as an alternative to chemical fungicides. Pseudomonas fluorescens SBW25 is one bacterial strain that has been demonstrated to promote plant growth by biocontrol of pathogenic fungi. To understand the mode of action of this bacterium, information regarding its localization and metabolic activity on plants is important. In this study, a gfp/luxAB-tagged derivative of P. fluorescens SBW25, expressing the green fluorescent protein (GFP) and bacterial luciferase, was monitored during colonization of wheat starting from seed inoculation. Since bacterial luciferase is dependent on cellular energy reserves for phenotypic expression, metabolically active cells were detected using this marker. In contrast, the stable GFP fluorescence phenotype was used to detect the cells independently of their metabolic status. The combination of these two markers enabled P. fluorescens SBW25 cells to be monitored on wheat plants to determine their specific location and metabolic activity. Studies on homogenized wheat plant parts demonstrated that the seed was the preferred location of P. fluorescens SBW25 during the 65-day time period studied, but the leaves and roots were also colonized. Interestingly, the bacteria were also found to be metabolically active on all plant parts examined. In situ localization of P. fluorescens SBW25 using a combination of different microscopic techniques confirmed the preference for the cells to colonize specific regions of the seed. We speculate that the colonization pattern of P. fluorescens SBW25 can be linked to the mechanism of protection of plants from fungal infection.     

Mycolytic effect of fluorescent Pseudomonas in biocontrolling of fungal phytopathogenic Curvularia lunata,Fusarium oxysporum,Alternaria padwickii and Rhizoctonia solani

About 50 bacterial strains, each of Pseudomonas fluorescens, from different rhizospheric soil of different plants were screened for antagonistic activity against Curvularia lunata, Fusarium oxysporum, Alternaria padwickii, Rhizoctonia solani causing black kernel, kernel spotting, root rots, stackburn and sheath blight diseases of rice (Oryza sativa L.). Out of the 50 isolates, 15 isolates were found to be effective in lysing the cell wall of the above-mentioned putative pathogens tested in vitro. These Pseudomonas isolates produced mycolytic enzymes, viz. β-1,3-glucanases, β-1,4-glucanases and lipases. P. fluorescens PAK1 and PAK12 among the strains were more effective for the production of these enzymes while PAK12 produce good level of β-1,3-glucanases, β-1,4-glucanases and lipases against tested fungal pathogens. These findings demonstrate a mechanism of antagonism by P. fluorescens against different fungal plant pathogens.     

Analysis of the draft genome of Pseudomonas fluorescens ATCC17400 indicates a capacity to take up iron from a wide range of sources,including different exogenous pyoverdines

All fluorescent pseudomonads (Pseudomonas aeruginosa, P. putida, P. fluorescens, P. syringae and others) are known to produce the high-affinity peptidic yellow-green fluorescent siderophore pyoverdine. These siderophores have peptide chains that are quite diverse and more than 50 pyoverdine structures have been elucidated. In the majority of the cases, a Pseudomonas species is also able to produce a second siderophore of lower affinity for iron. Pseudomonas fluorescens ATCC 17400 has been shown to produce a unique second siderophore, (thio)quinolobactin, which has an antimicrobial activity against the phytopathogenic Oomycete Pythium debaryanum. We show that this strain has the capacity to utilize 16 different pyoverdines, suggesting the presence of several ferripyoverdine receptors. Analysis of the draft genome of P. fluorescens ATCC 17400 confirmed the presence of 55 TonB-dependent receptors, the largest so far for Pseudomonas, among which 15 are predicted to be ferripyoverdine receptors (Fpv). Phylogenetic analysis revealed the presence of two different clades containing ferripyoverdine receptors, with sequences similar to the P. aeruginosa type II FpvA forming a separate cluster. Among the other receptors we confirmed the presence of the QbsI (thio)quinolobactin receptor, an ferri-achromobactin and an ornicorrugatin receptor, several catecholate and four putative heme receptors. Twenty five of the receptors genes were found to be associated with genes encoding extracytoplasmic sigma factors (ECF σ) and transmembrane anti-σ sensors.     

Recovery of Pseudomonas spp. from chronically fuel oil-polluted soils in Jordan and the study of their capability to degrade short chain alkanes

Enumeration of bacteria and recovery of the dominant species have been conducted for soils contaminated with fuel spills for more than 10 years at seven gas stations in Jordan. Bacterial counts of these polluted soils ranged between 0.68 × 10⁸ and 32.8 × 10⁸ c.f.u./g soil with two different bacterial colony types recovered on agar plates. Phenotypic examination of the recovered bacteria revealed that they belonged mainly to the genus Pseudomonas and was represented by the following species: P. acidovorans, P. putrefaciens, P. cepacia, P. vesicularis and P. fluorescens. The ability of these bacteria to grow on hexane or heptane was revealed by a colorimetric test. Action of five Pseudomonas spp.: Pseudomonas putrefaciens, P. cepacia, P. acidovorans, P. vesicularis and P. fluorescens and Rhodococcus erythropolis on 0.1% (v/v) hexane or heptane was followed at 1, 2, 6 and 12 h. Pseudomonas putrefaciens, P. cepacia and P. acidovorans were capable of degrading both hydrocarbons as indicated by the yellow colour formation (positive reaction); however, P. vesicularis and P. fluorescens showed no such capability.