Revised structures of the pyoverdins from Pseudomonas putida CFBP 2461 and from Pseudomonas fluorescens CFBP 2392

Several suggestions for structures of the siderophores (pyoverdins) from Pseudomonas spp. can be found in the literature which are based on a FAB mass spectrometric analysis only. Availability of two original strains of two Pseudomonas spp. allowed to re-investigate the structure of their pyoverdins. In both cases the amino acid sequence had to be corrected. In addition, d- and l-amino acids could be identified and located in the peptide chain. The knowledge of the correct structures is important in view of an ongoing study to establish relationships between the nature of the peptide chains of pyoverdins and their recognition by outer membrane proteins.     

Fluorene Cometabolism by Rhodococcus rhodochrous and Pseudomonas fluorescens Cultures

The transformation of fluorene by Rhodococcus rhodochrous strain 172 grown on sucrose and Pseudomonas fluorescens strain 26K grown on glycerol was studied as a function of the substrate concentration and the growth phase. Under certain cultivation conditions, fluorene was completely consumed from the medium. The specific transformation rate of fluorene was considerably higher when it was transformed in the presence of the cosubstrates than when it served as the sole carbon source. An approach to the evaluation of the specific transformation rate of fluorene during batch cultivations is proposed.     

Induction of polymyxin resistance in Pseudomonas fluorescens by phosphate limitation

Shift of Pseudomonas fluorescens NCMB 129 from a phosphate rich into a phosphate limited medium results in a reduction of the membrane phospholipids phosphatidylethanolamine, phosphatidylglycerol and cardiolipin. Concomitantly a positively charged ornithine amide lipid is synthesized. The gradual increase of this lipid is paralleled by an increasing resistance to polymyxin B. The binding capacities of intact cells, and isolated inner and outer membranes for the antibiotic are reduced in the resistant organisms. It is discussed that the observed effect could be circumstantial evidence that the positively charged polymyxin B needs negatively charged receptors in biological membranes in order to exert its antibiotic activity.List of Abbreviations PE phosphatidylethanolamine - PG phosphatidylglycerol - CL cardiolipin - PX polymyxin B     

Effect of temperature on diauxic growth with glucose and organic acids in Pseudomonas fluorescens

Growth of Pseudomonas fluorescens in batch culture with glucose and organic acids resulted in typical diauxic responses at 30° C but no detectable diauxic lag at 5° C.At 30° C, organic acids were preferentially utilized during the first growth phase. Glucose utilization was delayed unitl onset of the second growth phase. Systems involved in direct uptake and catabolism of glucose responded in a manner compatible with respression by malate and/or its metabolites and induction by glucose and/or its metabolites. The oxidative non-phosphorylated pathway, through gluconate and 2-ketogluconate (2-KG) as intermediates, was not induced during either growth phase.At 5° C, growth with glucose and organic acids was biphasic but without diauxic lag. Organic acids were preferentially utilized during the first growth phase. Although carbon from glucose was not fully catabolized until onset of the second growth phase, glucose was oxidized to and accumulated extracellularly as gluconate and 2-KG during the first growth phase. No significant repression of glucose-catabolizing enzymes was observed during growth with organic acids in the presence of glucose. However, uptake activities for gluconate and 2-KG did not increase significantly until onset of the second growth phase.Thus, at low temperatures, psychrotrophic P. fluorescens oxidized glucose to extracellular 2-KG, while growing on preferred carbon sources. The 2-KG was then catabolized after depletion of the organic acid.     

Extracellular Protease as a Reversible Adhesion Regulator in Pseudomonas fluorescens

The investigation of growth dynamics and protein content in a batch Pseudomonas fluorescens culture grown in a synthetic medium with glucose as the sole source of carbon and energy showed that cells reversibly adhere to the walls of the cultivation flask during the first 2–3 h of growth. Over this time period, the total protein content of free and bound cells increased exponentially at a rate of 0.25 h^–1, the fraction of proteins in cells being almost the same (60–70%). The protein content in the medium increased from 3 to 50 mg/l, reaching about 30% of the total protein of the culture. The addition of the exponential culture liquid filtrate to the medium together with the inoculum led to the complete inhibition of cell adhesion and a drastic activation of proteolysis, with a concurrent release of more than 80% of cellular proteins into the medium. After 3–5 h of growth, the concentration of extracellular proteins decreased to the control level. Exogenously added proteinase K inhibited cell adhesion, the effect being more pronounced for R-type than for S-type cells. The hypothesis is discussed that the short-term reversible adhesion of cells is regulated with the involvement of a mixture of hydrocarbons, which inactivate the functional activity of bacterial adhesins, and proteases, which digest these adhesins.     

Utilization of benzylpenicillin as carbon,nitrogen and energy source by a Pseudomonas fluorescens strain

A bacterium which utilizes benzylpenicillin as carbon, nitrogen and energy source was isolated from a lake sediment. The organism was identified as a strain of Pseudomonas fluorescens with a GC content of 59.71 Mol %. After growth of the organism on a mineral salts medium containing benzylpenicillin, the derivatives benzylpenicilloic acid, benzylpenilloic acid and benzylpenicillenic acid were found in culture media. There was no indication that the phenylacetate side chain of benzylpenicillin is decomposed. In uninoculated culture media benzylpenicillin, benzylpenicilloic acid and benzylpenicillenic acid were demonstrable. The following compounds were found to be absent from inoculated or uninoculated culture fluids: d-penicillamine, l-valine, l-cysteine, benzylpenillic acid and 6-aminopenicillanic acid. The organism possesses penicillinase. Penicillin acylase was not demonstrable. The reaction product of penicillinase, benzylpenicilloic acid, supports only little growth. There is no growth on 6-aminopenicillanic acid with or without NH₄Cl. Relatively little growth occurs on 6-aminopenicillanic acid in the presence of phenylacetic acid.The data indicate that the nucleus of the benzylpenicillin molecule is utilized as carbon, nitrogen and energy source. During growth a part of the substrate is destroyed into scarcely usable benzylpenicilloic acid; hereby the antibiotic is detoxified.Abbreviations TLC thin-layer chromatography - DNPH 2,4-dinitrophenylhydrazine     

The pyoverdine from Pseudomonas chlororaphis D-TR133 showing mutual acceptance with the pyoverdine of Pseudomonas fluorescens CHA0

From Pseudomonas chlororaphis D-TR133 a pyoverdine was isolated and its primary structure were elucidated by spectroscopic methods and degradation reactions. Despite some structural differences, its Fe(III) complex and that of the pyoverdine from Pseudomonas fluorescens CHA0 were taken up by either strain with a high rate. This is explained by a structural similarity between the two pyoverdines which were shown to differ in their structures only by the replacement of Lys by Ala in the C-terminal part of the molecules. An unexpected feature is that the main pyoverdine of P. chlororaphis D-TR133 is accompanied by a minor one where specifically one Ala is replaced by Gly. So far amino acid variations in the peptide chain of pyoverdines produced by a given strain had not been observed amongst the producers of the about fifty pyoverdines reported in the literature.     

Quorum-sensing system influences root colonization and biological control ability in Pseudomonas fluorescens 2P24

Pseudomonas fluorescens 2P24 is a biocontrol agent isolated from a wheat take-all decline soil in China. This strain produces several antifungal compounds, such as 2,4-diacetylphloroglucinol (2,4-DAPG), hydrogen cyanide and siderophore(s). Our recent work revealed that strain 2P24 employs a quorum-sensing system to regulate its biocontrol activity. In this study, we identified a quorum-sensing system consisting of PcoR and PcoI of the LuxR–LuxI family from strain 2P24. Deletion of pcoI from 2P24 abolishes the production of the quorum-sensing signals, but does not detectably affect the production of antifungal metabolites. However, the mutant is significantly defective in biofilm formation, colonization on wheat rhizosphere and biocontrol ability against wheat take-all, whilst complementation of pcoI restores the biocontrol activity to the wild-type level. Our data indicate that quorum sensing is involved in regulation of biocontrol activity in P. fluorescens 2P24.     

Horizontal and vertical movement of Pseudomonas fluorescens toward exudate of Macrophomina phaseolina in soil: influence of motility and soil properties

The role of motility and cell surface hydrophobicity in transport and dispersal of Pseudomonas fluorescens strains LAM1-hydrophilic, LAM2-hydrophobic and LAM(NM) (non-motile mutant of LAM2) under different soil conditions was studied. Maximum adhesion was recorded for LAM2 in clay loam (70%), followed by sandy loam (68%) and sandy soil (40%). Vertical migration of P fluorescens isolates in soils was recorded at 5 and 25 cm flow of wafer or M. phaseolina exudate. In all the treatments, LAM1 exhibited maximum migration followed, by LAM2 and LAM(NM). The rate of migration of such isolates was lowered in water irrigated soils compared to those irrigated with M. phaseolina exudate. In sandy soil, cells of LAM1 migrated up to 13 cm in comparison to LAM2 (11 cm) and LAN(NM) (9 cm) at 5 cm flow of fungal exudate. Population of LAM1, LAM2 and LAM(NM) was 5.7, 5.68 and 5.61 log cfu g(-1) soil at 1 cm depth, but it decreased to 2.56, 2.21 and 1.99 log cfu during migration up to 11 cm in sandy soil at 5 cm flow of fungal exudate. Greater motility was observed in sandy soil irrigated with water or fungal exudate, followed by sandy loam and clay loam. In general, filtration coefficient (lambda) of P. fluorescens was higher in soils irrigated with 5 cm of water or exudate than with 25 cm of irrigation. The horizontal movement of P. fluorescens strains in sandy soil adjusted at different psi m showed marked reduction with decrease in psi m. The non-motile LAN(NM) did not show chemotactic response and migrated up to a maximum of 3 mm in saturated soils (0 kPa). After 96 h, LAM1 and LAM2 migrated upto 35 and 29 mm respectively in sandy soil. Motile isolates had significantly greater colonization of M. phaseolina sclerotia over the non-motile mutant.     

Effect of Pseudomonas fluorescens on the root exudates of two tomato mutants differently sensitive to Fe chlorosis

Plants with different Fe-mobilization properties are known to differ in the amount and kind of Fe-reducing and Fe-chelating compounds exuded by their roots. Although rhizosphere bacteria are known to affect the exudation of organic compounds by the plant roots, their effect on the root exudates of plants differing in Fe-mobilization properties is not known. We studied the effect of Pseudomonas fluorescens, on the exudation of sugars and organic and amino acids by roots of an iron chlorosis-resistant (T3238FER) and a chlorosis-susceptible (T3238fer) tomato mutant. Under sterile conditions two tomato mutants grew equally well and did not differ in the total amount of sugars and organic acid exuded by their roots. More amino acids, however, were exuded by the roots of T3238FER than T323fer. Mutants differed in the amount of oxalic acid and the amino acids Ala, Asp, Gaba, Gln, Gly, His, Hyl, Ile, Leu, Lys, Phe, Pro, and Val exuded by their roots into sterile rooting media. Addition of P. fluorescens to the rooting medium did not affect the growth of T3238FER but stimulated the root growth of chlorosis-susceptible T3238fer, reduced the amounts of glucose, arabinose and fructose but increased the amount of sucrose, reduced the amounts of fumaric, malic and oxalic acid but increased the amounts of citric and succinic acid in the rooting media of both mutants. P. fluorescens resulted in the following changes in the amino acids in the rooting media: reduced the amounts of Gly, Leu, and Lys in T3238FER, and of Asp, Gln, Hyp, and Ile in T3238fer, and increased the amounts of Cys, Glu, His, Hyp, Ile, Phe and Tyr in T3238FER and of Ala, Glu, His, Phe, and Ser in T323fer—in cases more than 40-fold. These differential effects of P. fluorescens in altering the pattern of organic and amino acids compounds with some Fe-chelating properties detected in the rooting medium of these two mutants may indicate that the differences in Fe-chlorosis susceptibility of these tomato mutants may be the result of, or modified by, the interactions between plant roots and rhizosphere microorganisms. We postulate that the Fe-chlorosis susceptibility in plants may be the product of the interactions between soil microorganisms and plant roots, and may not be solely related to the plant per se.     

Proteomics of a plant growth-promoting rhizobacterium, Pseudomonas fluorescens MSP-393, subjected to salt shock

Summary Pseudomonas fluorescens MSP-393, a plant growth-promoting rhizobacterium is an efficient biocontrol agent in rice grown in saline soils of coastal ecosystems. To understand the mechanism of salt tolerance, proteome analysis of the bacterium was carried out employing two-dimensional gel electrophoresis and MALDI-TOF. This technique was used to investigate the regulation of gene product expression of P. fluorescens MSP-393 grown under high osmolarity and used peptide mass fingerprinting and in silico investigation to identify those proteins with altered expression. Among them 15 were assigned to proteins with known functions. Their roles in response to salt stress are discussed.     

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 substrate specificity permitting the oxidation of a suite of polycyclic aromatic and heterocyclic substrates.     

Engineering of histidine tail in the N-terminal region of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens

The pobA of Pseudomonas florescens IFO14160 encoding a p-hydroxybenzoate hydroxylase (PHBH) was cloned, sequenced, and over-expressed in Escherichia coli. To facilitate the purification of PHBH, a fully active, tagged enzyme was constructed by engineering a two-, three-, or six-histidine tail in the N-terminal region (H2-, H3-, or H6-PHBH), or a six-histidine tail in the C-terminal region of the PHBH. The six-histidine tail in the C-terminal region of the enzyme could not be expressed with activity, while the other polyhistidine tails in the N-terminal region of the enzyme were highly active. However, the resulting H6-PHBH could not be purified by Ni-NTA chromatography because the H6-PHBH was so strongly bound to the supports that it could be eluted only after a significant change in conditions. On the other hand, H2-PHBH did not bind tightly to the Ni-chelate support. H3-PHBH was purified from the crude extract in a single step by using the optimized length and location of the polyhistidine tail in the enzyme.     

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     

Growth and Adhesion of Pseudomonas fluorescens in a Batch Culture: A Kinetic Analysis of the Action of Extracellular Antiadhesins

This work tests 6 hypothetical models simulating the growth, respiration, and adhesion of cells to the walls of the cultivation flask. All the models postulate the synthesis of antiadhesins (AAs), i.e., extracellular metabolites decreasing the degree of cell adhesion. The models have the following distinguishing features: (model 1) the blocking of sorption centers on the glass walls by antiadhesins (the competitive inhibition of adhesion); (model 2) the noncompetitive inhibition of adhesion; (model 3) the accelerated release of bound cells; (model 4) a combination of models 1 and 3; (model 5) a combination of models 1 and 3 with a delay; (model 6) a combined action of two AAs, one of which, AA₁, inhibits cell adhesion, and the other, AA₂ (its synthesis is induced when the concentration of AA₁ reaches a threshold level), stimulates the detachment of bound cells. Model 6 fits the relevant experimental data best. The delay effect is relatively small. The sigmoid character of the curve showing cell adhesion as a function of the antiadhesin concentration implies the existence of a strong cooperative effect in the adhesion inhibition. The models proposed satisfactorily simulate the growth, respiration, and adhesion of cells and AA synthesis in a batch bacterial culture grown either in a fresh nutrient medium or in the medium supplemented with the filtrate of a mature culture of the same species.     

Structure and Properties of the Lipopolysaccharide of Pseudomonas fluorescens IMV 2366 (Biovar III)

The lipopolysaccharide (LPS) preparation isolated from the bacterial mass of Pseudomonas fluorescens IMV 2366 (biovar III) by Westphal's method and purified by repeated ultracentrifugation contained S- and R-forms of molecules. The structural components of the LPS molecule—lipid A, core oligosaccharide, and O-specific polysaccharide—were obtained in the individual state and characterized. The main components of the lipid A hydrophobic moiety were 3-hydoxydecanoic, 2-hydroxydodecanoic, 3-hydroxydodecanoic, dodecanoic, and hexadecanoic fatty acids. Glucosamine, phosphoethanolamine, and phosphorus were identified as the components of the lipid A hydrophilic moiety. Rhamnose, glucose, galactose, glucosamine, galactosamine, alanine, phosphoethanolamine, phosphorus, and 2-keto-3-deoxyoctulosonic acid (KDO), as well as 2-amino-2,6-dideoxygalactose (FucN) and 3-amino-3,6-dideoxyglucose (Qui3N), were revealed in the composition of the core oligosaccharide fractions. O-specific polysaccharide chains were composed of repeating trisaccharide units consisting of residues of L-rhamnose (L-Rha), 2-acetamido-2,6-dideoxy-D-galactose (D-FucNAc), and 3-acylamido-3,6-dideoxy-D-glucose (D-Qui3NAcyl), where Acyl = 3-hydroxy-2,3-dimethyl-5-hydroxyprolyl. Neither double immunodiffusion in agar not the immunoenzymatic assay revealed serological relations between the strain studied and the P. fluorescens strains studied earlier.     

Chromate resistance and reduction in Pseudomonas fluorescens strain LB300

Pseudomonas fluorescens LB300 is a chromateresistant strain isolated from chromium-contaminated river sediment. Chromate resistance is conferred by the plasmid pLHB1. Strain LB300 grew in minimal salts medium with as much as 1000 g of K₂CrO₄ ml^–1, and actively reduced chromate to Cr(III) while growing aerobically on a variety of substrates. Chromate was also reduced during anaerobic growth on acetate, the chromate serving as terminal electron acceptor. P. fluorescens LB303, a plasmidless, chromatesensitive variant of P. fluorescens LB300, did not grow in minimal salts medium with more than 10 g of K₂CrO₄ ml^–1. However, resting cells of strain LB303 grown without chromate reduced chromate as well as strain LB300 cells grown under the same conditions. Furthermore, resting cells of chromate-sensitive Pseudomonas putida strain AC10, also catalyzed chromate reduction. Evidently chromate resistance and chromate reduction in these organisms are unrelated. Comparison of the rates of chromate reduction by chromate grown cells and cells grown without chromate indicated that the chromate reductase activity is constitutive. Studies with cell-free extracts show that the reductase is membrane-associated and can mediate the transfer of electrons from NADH to chromate.     

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.     

Some properties of thePseudomonas fluorescens adhesin and antiadhesin

Some properties of the adhesion-modifying factors ofPseudomonas fluorescens are described. Adhesin, which promotes the adhesion ofP. fluorescens cells, is a hydrophobic compound of a protein nature with a molecular mass of more than 10 kDa located either at the cell surface or in the medium. Antiadhesin, which suppresses the adhesion ofP. fluorescens cells, is a thermolabile hydrophobic compound of a nonprotein nature with a molecular mass of less than 3 kDa. Heating makes antiadhesin hydrophilic. The role of adhesin and antiadhesin in the adhesion and adaptation ofP. fluorescens cells is discussed.     

Biological properties of the wild rhizosphere strain Pseudomonas fluorescens 2137 and its derivatives marked with the gusA gene

he natural wild rhizosphere strain P. fluorescens 2137 was marked with the β-glucuronidase gene gusA. The introduction of this gene influenced the viability of the wild strain, as well as its certain physiological parameters, such as cultural characteristics, biochemical properties, and antagonistic activity against the phytopathogenic fungi Fusarium culmorum, F. oxysporum, F. graminearum, and Verticillum nigrescens. The gusA-marked derivative strains that deviate the least from the wild strain in biological properties can be used to monitor populations of P. fluorescens 2137 cells in the plant rhizosphere.