Exocellular phosphatidylethanolamine production and multiple-metal tolerance in Pseudomonas fluorescens

Abstract Pseudomonas fluorescens appeared to circumvent the challenge imposed by millimolar amounts of metals (5 mM Al³⁺, 5 mM Fe³⁺, 2 mM Ca²⁺, 1 mM Ga³⁺ and 3 mM Zn²⁺) by the formation of phosphatidylethanolamine. This lipid moiety constituted an important organic component of an insoluble gelatinous residue in which most of the test metals were immobilized at stationary phase of growth. Ultracentrifugation and dialysis experiments showed that the metals were associated with phosphatidylethanolamine from early stages of growth. Transmission electron microscopy revealed metal rich bodies in the cytoplasm prior to their secretion in the spent fluid. These results demonstrate a role of phosphatidylethanolamine in multiple-metal homeostasis.     

Oxalic acid production and aluminum tolerance in Pseudomonas fluorescens

13C NMR studies on intact cells from Al-stressed Pseudomonas fluorescens incubated with citric acid or Al-citrate yielded peaks at 158 and 166 ppm that were attributable to free and complexed oxalic acid, respectively. The presence of oxalic acid was further confirmed with the aid of oxalate oxidase. These peaks were not discernable in experiments performed with cells taken from control cultures. Enzymatic analyses of cell fractions showed the highest production of oxalic acid in the inner membrane fraction of Al-stressed cells incubated with glyoxylate. There was an eight-fold increase in the synthesis of oxalic acid in the inner membrane fraction from the Al-stressed cells compared to the control cells. Although oxalic acid production was observed when citrate, Al-citrate and isocitrate were utilized as substrates, the inner membrane fraction did not mediate the formation of oxalic acid from glycine/pyruvate, glycolic acid, oxaloacetate or ascorbate. These data suggest that the increased oxalic acid production in response to Al stress is effected via the oxidation of glyoxylate.     

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     

Influence of magnesium ions on biofilm formation by Pseudomonas fluorescens

Mg²⁺ can potentially influence bacterial adhesion directly through effects on electrostatic interactions and indirectly by affecting physiology-dependent attachment processes. However, the effects of Mg²⁺ on biofilm structure are largely unknown. In this study, Pseudomonas fluorescens was used to investigate the influence of Mg²⁺ concentration (0, 0.1 and 1.0 mM MgCl₂) on biofilm growth. Planktonic and attached cells were enumerated (based on DAPI staining) while biofilm structures were examined via confocal laser scanning microscopy and three-dimensional structures were reconstructed. Mg²⁺ concentration had no influence on growth of planktonic cells but, during biofilm formation, Mg²⁺ increased the abundance of attached cells. For attached cells, the influence of Mg²⁺ concentration changed over time, suggesting that the role of Mg²⁺ in bacterial attachment is complex and dynamic. Biofilm structures were heterogeneous and surface colonization and depth increased with increasing Mg²⁺ concentrations. Overall, for P. fluorescens, Mg²⁺ increased initial attachment and altered subsequent biofilm formation and structure.     

Aluminum tolerance in Pseudomonas fluorescens ATCC 13525: Involvement of a gelatinous lipid-rich residue

Abstract Pseudomonas fluorescens was found to grow in a mineral medium supplemented with up to 50 mM aluminum, complexed to citrate, the sole source of carbon. At stationary phase while virtually no diminution in cellular yield was observed in the presence of 1.0 mM aluminum, only 31% of bacterial yield was recorded in media with 50 mM aluminum. The decrease in soluble aluminum in the culture fluid was concomitant with the formation of a gelatinous residue. At later stages of growth, the trivalent metal was immobilized in this deposit. This bioprecipitate consisted of lipid moieties but was apparently devoid of proteins and carbohydrates. X-ray fluorescence spectroscopy and colorimetric assays also revealed the presence of aluminum and phosphorus. X-ray diffraction spectroscopy indicated that the biomineral was amorphous. Examination of the gelatinous residue by scanning electron microscopy and energy dispersive X-ray microanalysis aided in the identification of aluminum, oxygen and phosphorus rich irregular bodies that were associated with carbon containing thread-like structures.     

荧光假单胞菌生防机理的研究进展

荧光假单胞菌是植物根际促生细菌(Plant Growth Promoting Rhizobacteria,PGPR)具有分布广、数量多、营养需要简单、繁殖快、竞争定殖力强的特点。它们能通过产生多种次生代谢物及有效的根际定殖防治植物病害,成为植物生防控制的重要研究对象。主要论述了荧光假单胞菌对植物病害生物防治机理的研究进展。     

Inactivation of Salmonella serovars by Pseudomonas chlororaphis and Pseudomonas fluorescens strains on tomatoes

Salmonella enterica and its serovars have been associated with pathogen contamination of tomatoes with numerous outbreaks of salmonellosis. To improve food safety, pathogen control is of immediate concern. The aim of this research was to assess the populations of natural microflora (aerobic mesophilic bacteria, lactic acid bacteria, yeasts and moulds and Pseudomonas species) on tomatoes, and evaluate the efficacy of Pseudomonas fluorescens (Pf) and Pseudomonas chlororaphis (Pc) for inactivation of Salmonella on tomatoes. Microflora were determined on sanitised and unsanitised produce and enumerated on Plate Count Agar, de Man, Rogosa and Sharpe medium, Potato Dextrose Agar and Pseudomonas Agar F media. The efficacy of Pc and Pf for inactivation of S. enterica serovars Montevideo, Typhimurium and Poona was determined on spot-inoculated tomato stem scars. The effects of storage time on bacterial populations were also investigated. On unsanitised tomatoes, lactic acid bacteria, Pseudomonas sp., aerobic mesophilic bacteria and yeasts and moulds ranged from 3.31–4.84, 3.93–4.77, 4.09–4.80 and 3.83–4.67 log CFU/g of produce, respectively. The microflora were similar at 0 and 24 storage hours on sanitised produce. The suppression of Salmonella Montevideo by P. chlororaphis and P. fluorescens on tomatoes ranged from 0.51 to 2.00 log CFU/g of produce. On Salmonella Montevideo and S. Typhimurium, the suppressive effects ranged from 0.51 to 0.95 and 0.46 to 2.00 log CFU/g of produce, respectively. The pathogen suppressive effects may be attributed to competition ability of Pseudomonas relative to Salmonella strains. Pseudomonas strains may be effective against Salmonella strains as a post-harvest application, but strain synergy is required to optimise pathogen reductions.     

Adaptive landscapes in evolving populations of Pseudomonas fluorescens

The repeatability of adaptive evolution depends on the ruggedness of the underlying adaptive landscape. We contrasted the relative ruggedness of two adaptive landscapes by measuring the variance in fitness and metabolic phenotype within and among genetically distinct strains of Pseudomonas fluorescens in two environments differing only in the carbon source provided (glucose vs. xylose). Fitness increased in all lines, plateauing in one environment but not the other. The pattern of variance in fitness among replicate lines was unique to the selection environment; it increased over the course of the experiment in xylose but not in glucose. Metabolic phenotypes displayed two results: (1) populations adapted via changes that were distinctive to their selection environment, and (2) endpoint phenotypes were less variable in glucose than in xylose. These results indicate that although the response to selection is highly repeatable at the level of fitness, the underlying genetic routes taken were different for each environment and more variable in xylose. We suggest that this reflects a more rugged adaptive landscape in xylose compared to glucose. Our study demonstrates the utility of using replicate selection lines with different evolutionary starting points to try and quantify the relative ruggedness of adaptive landscapes.     

Influence of an arbuscular mycorrhizal fungus on Pseudomonas fluorescens DF57 in rhizosphere and hyphosphere soil

The influence of Glomus intraradices (BEG87) on Pseudomonas fluorescens DF57 in hyphosphere and rhizosphere soil was examined. Cucumis sativus (Aminex, F₁ hybrid) was grown in symbiosis with the arbuscular mycorrhizal fungus G. intraradices in PVC tubes, consisting of a central root compartment and two lateral root-free compartments. Two Tn 5 - lux AB-marked strains of P. fluorescens DF57 were used. Strain DF57-P2, which has an insertion of Tn 5::lux AB in a phosphate starvation-inducible locus, was used as a phosphate starvation reporter. Another lux -tagged strain DF57-40E7, which carries a constitutively expressed lux AB fusion, was used as control for strain DF57-P2 and for measuring the metabolic activity of P. fluorescens DF57. A strain of P. fluorescens DF57, which carries a constitutively expressed gfp gene, was used in studies of attachment between the bacteria and the hyphae. G. intraradices decreased the culturability of P. fluorescens DF57 significantly, both in rhizosphere and hyphosphere soil, whereas the total number of P. fluorescens DF57 measured by immunofluorescence microscopy was decreased in hyphosphere soil only. G. intraradices did not induce a phosphorus starvation response in P. fluorescens DF57, and the metabolic activity of the bacteria was not affected by the fungus after 48 h. P. fluorescens DF57 did not attach to G. intraradices hyphae and was not able to use the hyphae as carbon substrate. The negative effect of G. intraradices on culturability and on number of P. fluorescens DF57 in hyphosphere soil is discussed.     

荧光假单胞杆菌P13菌株对油菜化感作用的研究

通过化感试验研究荧光假单胞杆菌P13菌株对油菜种子萌发、幼苗生长的影响及其在油菜根际土壤和根部定植的能力。结果表明,P13菌株发酵液对油菜种子萌发没有明显的促进作用,稀释10倍的菌体发酵液处理种子与对照组无显著差异,而低浓度和高浓度都抑制种子萌发;田间试验发现P13菌株能促进植物幼苗生长,根长、苗高、干重和长1片叶子的株数均与对照组差异显著;1周内P13菌株在油菜根际土壤和根部定植良好,定植数量均达到107cfu/g以上。说明P13菌株可被开发为微生物菌剂,但在施用时不宜用作种子处理剂。     

Gallium toxicity and adaptation in Pseudomonas fluorescens

When cultured in a defined citrate medium supplemented with 1 mM gallium (III) Pseudomonas fluorescens ATCC 13525 experienced a lag phase of 40 h with no apparent diminution in cellular yield. Following initial uptake of the metal-ligand complex, gallium was secreted in the spent fluid. This lag phase was abolished either by inoculating the medium with gallium adapted cells or by inclusion of iron (III) (20 microM) in the growth medium. In the culture enriched with both gallium and iron (III), X-ray fluorescence spectra revealed a gradual decrease of gallium from the spent fluid as growth progressed. In a phosphate deficient medium, no cellular multiplication was observed in the presence of gallium. The inhibitory influence mediated by the trivalent metal was reversed by the addition of (20 microM) iron (III). Although bacterial growth was accompanied by an initial decrease in exocellular gallium, a marked increment in the concentration of this metal was observed in the spent fluid at stationary phase of growth. Citrate was not detected in the exocellular fluid at cessation of bacterial multiplication. Electrophoretic analyses revealed numerous variations in the cytoplasmic protein profiles of the control and metal stressed cells. Gallium induced the syntheses of polypeptides with apparent molecular masses of 89 kDa, 50 kDa, 39 kDa, 26 kDa and 12 kDa.     

Ethylbenzene degradation by Pseudomonas fluorescens strain CA-4

Abstract Pseudomonas fluorescens strain CA-4 is a bioreactor isolate capable of ethylbenzene degradation. Transposon mutagenesis and enzyme assays have been performed which allow us to propose the ethylbenzene degradative pathway in operation in this strain. Ethylbenzene is initially converted to 2-phenylethanol. This is degraded to phenylacetaldehyde and then to phenylacetic acid. The major inducer of the pathway is ethylbenzene itself. The pathway is regulated by the presence of non-aromatic carbon sources. Oxidation of ethylbenzene is repressed by glutamate, but not by citrate or glucose. A clone from a chromosomal library has been found to complement a mutant deficient in the ability to convert ethylbenzene to 2-phenylethanol.     

Nitrite inhibition of denitrification by Pseudomonas fluorescens

Using a pure culture of Pseudomonas fluorescens as a model system nitrite inhibition of denitrification was studies. A mineral media with acetate and nitrate as sole electron donor and acceptor, respectively, was used. Results obtained in continuous stirred-tank reactors (CSTR) operated at pH values between 6.6 and 7.8 showed that growth inhibition depended only on the nitrite undissociated fraction concentration (nitrous acid). A mathematical model to describe this dependence is put forward. The maximum nitrous acid concentration compatible with cell growth and denitrification activity was found to be 66 mug N/L. Denitrification activity was partially associated with growth, as described by the Luedeking-Piret equation. However, when the freshly inoculated reactor was operated discontinuosly, nitrite accumulation caused growth uncoupling from denitrification activity. The authors suggest that these results can be interpreted considering that (a) nitrous acid acts as a proton uncoupler; and (b) cultures continuoulsy exposed to nitrous acid prevent the uncoupling effect but not the growth inhibition. Examination of the growth dependence on nitrite concentration at pH 7.0 showed that adapted cultures (grown on CSTR) are less sensitive to nitrous acid inhibition than the ones cultivated in batch. (c) 1995 John Wiley & Sons, Inc.     

Growth of Pseudomonas fluorescens in modified atmosphere packaged tofu

Aims: The objective was to study the growth of Pseudomonas in a food product (tofu) where it typically occurs as a spoilage organism, and when this product is stored under modified atmosphere. Methods and Results: A Pseudomonas strain was isolated from the endogenous microflora of tofu. Tofu was inoculated with the strain, packaged in different gas conditions (air, 100% N₂, 30% CO₂/70% N₂ or 100% CO₂) and stored under refrigerated conditions. Microbial loads and the headspace gas composition were monitored during storage. Conclusions: The strain was capable of growing in atmospheres containing no or limited amounts of oxygen and increased amounts of carbon dioxide. Even when 100% CO₂ was used, growth could not be inhibited completely. Significance and Impact of Study: In contrast to the general characteristics of the genus Pseudomonas (strictly aerobic, highly sensitive to CO₂), it should not be expected in the food industry that removing oxygen from the food package and increasing the carbon dioxide content, combined with cold storage, will easily avoid spoilage by Pseudomonas species. Guarantee of hygienic standards and combination of strategies with other microbial growth inhibiting measures should be implemented.     

一株荧光假单胞杆菌的分离鉴定与反硝化特性

【目的】从污水厂的活性污泥中获得一株高效反硝化细菌。【方法】采用低温驯化,进行初筛、复筛选取一株反硝化活性最高的菌株,命名为L2,通过形态学、生理生化特征及16S r RNA基因序列分析研究其分类地位,系统研究理化因素对该菌株反硝化性能的影响。【结果】菌株在低温条件下能够稳定高效地进行反硝化,鉴定该菌株为荧光假单胞杆菌(Pseudomonas fluorescens),其反硝化最适接种量为10%,温度为20°C,p H为7.0,盐浓度为0.5%,碳源为葡萄糖,C/N为5.0,能够耐受较高初始硝态氮浓度。【结论】菌株L2是一株耐低温、耐高浓度初始硝态氮、耐低C/N、兼性厌氧、高效反硝化的荧光假单胞杆菌。     

荧假单胞杆菌化感作用的初步研究

1　引　　言化感作用 (Allelopathy)是指一种植物或微生物通过产生化学物质而对其它生物产生的直接或间接的刺激或抑制作用[7] .虽然有关高等植物之间化感作用的研究已有大量报道 ,但微生物对高等植物的化感作用研究报道却较少 ,尤其是细菌在生态系统中的化感作用往往被忽视[1] .荧光假单胞杆菌 (P .fluorescens)是定殖于植物根际的优势细菌种群 ,此类细菌以其分布广、适应能力强、繁殖速度快、易于人工培养等特点 ,成为最具生防潜力和应用价值的生防菌[5] .对陕西农田土壤有益微生物的筛选研究中发现 ,一株荧光假单胞杆菌培养液对番茄灰霉…     

Influence of aluminum on motility and swarming of Pseudomonas sp. and Arthrobacter sp.

The effect of Al on the motility and the swarming of Pseudomonas sp. and Arthrobacter sp. was investigated. pH calibration curves were determined in semi-solid agar which enabled us to distinguish between the effects of aluminum and protons. Motility of the bacteria was distinctly influenced by Al but this effect could be attributed to the increased acidity accompanying Al addition. The swarming of Pseudomonas sp. is reported for the first time. The swarming ability decreased with both Al and HCl addition to the medium but the obstruction due to aluminum was significantly stronger than the influence of HCl.