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.     

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.     

Four genes from Pseudomonas fluorescens that encode the biosynthesis of pyrrolnitrin.

Pyrrolnitrin is a secondary metabolite of Pseudomonas and Burkholderia sp. strains with strong antifungal activity. Production of pyrrolnitrin has been correlated with the ability of some bacteria to control plant diseases caused by fungal pathogens, including the damping-off pathogen Rhizoctonia solani. Pseudomonas fluorescens BL915 has been reported to produce pyrrolnitrin and to be an effective biocontrol agent for this pathogen. We have isolated a 32-kb genomic DNA fragment from this strain that contains genes involved in the biosynthesis of pyrrolnitrin. Marker-exchange mutagenesis of this DNA with Tn5 revealed the presence of a 6.2-kb region that contains genes required for the synthesis of pyrrolnitrin. The nucleotide sequence of the 6.2-kb region was determined and found to contain a cluster of four genes that are required for the production of pyrrolnitrin. Deletion mutations in any of the four genes resulted in a pyrrolnitrin-nonproducing phenotype. The putative coding sequences of the four individual genes were cloned by PCR and fused to the tac promoter from Escherichia coli. In each case, the appropriate tac promoter-pyrrolnitrin gene fusion was shown to complement the pyrrolnitrin-negative phenotype of the corresponding deletion mutant. Transfer of the four gene cluster to E. coli resulted in the production of pyrrolnitrin by this organism, thereby demonstrating that the four genes are sufficient for the production of this metabolite and represent all of the genes required to encode the pathway for pyrrolnitrin biosynthesis.     

Pyrimidine ribonucleoside catabolism in Pseudomonas fluorescens biotype A

The pyrimidine ribonucleosides uridine or cytidine were shown to serve as a source of nitrogen or carbon for the growth of Pseudomonas fluorescens strain A126. After incubation of either pyrimidine ribonucleoside with extracts of this strain, the resultant catabolic products were detected by thin-layer chromatography. It was found that pyrimidine ribonucleoside catabolism in this pseudomonad involved the enzymes nucleoside hydrolase and cytosine deaminase. The specific activities of both these enzymes could be influenced by the nitrogen or carbon source present in the medium.     

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.     

Biological control of take-all disease by isolates of Pseudomonas fluorescens and biosynthesis of silver nanoparticles by the culture supernatant of Pseudomonas fluorescens CHA0

Plant diseases are among the main constraints affecting the production and productivity of crops both in terms of quality and quantity. Use of chemicals continues to be the major tactic to mitigate the menace of crop diseases. However, because of the environmental concerns, health conscious attitude of human beings and other hazards associated with the use of chemicals, use of bio agents to suppress the disease-causing activity of plant pathogens is gaining importance. With the emergence and increase of microbial organisms resistant to multiple antibiotics, and the continuing emphasis on health-care costs, many researchers have tried to develop new and effective antimicrobial reagents that do not stimulate resistance and are less expensive. Nanoscale materials have emerged as novel antimicrobial agents owing to their high surface area to volume ratio and the unique chemical and physical properties, which increases their contact with microbes and their ability to permeate cells. Since silver displays multiple modes of inhibitory action to micro-organisms, it may be used for controlling various plant pathogens in a relatively safer way compared to synthetic fungicides. Development of reliable and eco-friendly processes for synthesis of metallic nanoparticles is an important step in the field of application of nanotechnology. One of the options to achieve this objective is to use synthesis of nanoparticles of silver by reduction of aqueous Ag⁺ ions with the culture supernatant of Pseudomonas fluorescens CHA0. In this study, P. fluorescens CHA0 that has a medium impact on Gaeumannomyces graminis var. tritici was selected. Then, P. fluorescens CHA0 was used for the synthesis of silver nanoparticles. The morphology of the nanoparticles was characterised by Transmission Electron Microscopy and UV–vis spectroscopy. The silver nanoparticles of approximate size 50 nm were observed. The process of reduction is extracellular which makes it an easier method for the synthesis of silver nanoparticles.     

Inducible catalase in Pseudomonas fluorescens

The catalase activity of a non-proliferating suspension of Pseudomonas fluorescens doubled after six hours incubation in a 50 mM phosphate buffer medium (pH 7.3). The same effect was observed in a peptone medium. The increased activity was due to induced enzyme synthesis, and not to activation of preexisting catalase. Induced catalase was separated by electrophoresis from deuterium labelled constitutive catalase. The enzyme was also induced under anaerobic conditions in phosphate buffer or in culture when nitrate was supplied as an electron acceptor. Induction was considerably increased by the addition of various nucleotides and amino acids to the incubation medium.     

荧光假单胞菌2P24中retS对抗生素2，4-二乙酰基间苯三酚合成的影响

假单胞菌中RetS是一个位于膜上的感应激酶,对多种基因的表达都有调控作用.在铜绿假单胞菌中,RetS可以与另一个感应激酶GacS直接互作,并抑制GacS的磷酸化.[目的]本文利用遗传学方法研究了荧光假单胞菌2P24中RetS对抗生素2,4-二乙酰基间苯三酚(2,4-DAPG)合成的影响,并对其可能的调控机制进行了初步探索.[方法]利用高压液相色谱法(HPLC)检测2P24及其衍生菌株中2,4-DAPG的产量.将Gac/Rsm 信号途径中小RNA及调控蛋白的转录报告质粒转入到菌株2P24及其retS突变菌株中,检查RetS对以上基因转录表达的影响.[结果]菌株2P24中缺失retS后未知红色素和抗生素2,4-DAPG的产量较野生型均明显升高.进一步试验表明,RetS转录水平负调控小RNA RsmX和RsmZ的表达,这说明RetS可在转录后水平影响2,4-DAPG的合成.然而,同时缺失retS和gacS或同时缺失retS和gacA之后,由retS单基因缺失所造成的未知红色素和2,4-DAPG合成量升高、小RNA转录表达增强等性状消失,而与gacS或gacA单基因缺失突变体的表型一致.[结论]以上结果说明菌株2P24中RetS是2,4-DAPG及未知红色素合成的负调控因子,并且RetS对2,4-DAPG及未知红色素合成的调控依赖于Gac/Rsm信号传递路径.     

Fatty acid composition of lipid A in Pseudomonas fluorescens

The fatty acid composition of lipid A was studied using gas-liquid chromatography (GLC) and GLC-mass spectrometry in Pseudomonas fluorescens strains of biovars A, B, C, i, F and G, the type strain ATCC 13525 (biovar A) inclusive. The following fatty acids were identified as predominant in the composition of lipid A in the strains representing biovars A, B, C, i, F and G: 3-hydroxydecanoic (3-OH C10:0), 2-hydroxydodecanoic (2-OH C12:0), 3-hydroxydodecanoic (3-OH C12:0), dodecanoic (C12:0), hexadecanoic (C16:0), octadecanoic (C18:0), hexadecenoic (C16:1) and octadecenoic (C18:1) acids. Lipid A of a biovar G strain differed noticeably from other strains in its fatty acid composition. Its main components were as follows: 3-hydroxytetradecanoic (3-OH C14:0), 3-hydroxypentadecanoic (3-OH C15:0) and dodecanoic (C12:0) fatty acids. The coefficients of similarity were determined for lipid A specimens isolated from the studied strains of P. fluorescens by calculating their fatty acid composition with a computer.     

A new metalloproteinase from Pseudomonas fluorescens biotype I

Isolation and purification of a metalloproteinase from Pseudomonas fluorescens Biotype I are described. The molecular mass of the enzyme is 46 kDa, its isoelectric point is 8.1, its activity is trypsin-like. The amino-acid composition of the single chain protein is given. One molecule of the enzyme contains 1 atom of zinc and 9 atoms of calcium.     

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…     

Cadmium-regulated gene fusions in Pseudomonas fluorescens

To study the mechanisms soil bacteria use to cope with elevated concentrations of heavy metals in the environment, a mutagenesis with the lacZ-based reporter gene transposon Tn5B20 was performed. Random gene fusions in the genome of the common soil bacterium Pseudomonas fluorescens strain ATCC 13525 were used to create a bank of 5,000 P. fluorescens mutants. This mutant bank was screened for differential gene expression in the presence of the toxic metal cadmium. Fourteen mutants were identified that responded with increased or reduced gene expression to the presence of cadmium. The mutants were characterized with respect to their metal-dependent gene expression and their metal tolerance. Half the identified mutants reacted with differential gene expression specifically to the metal cadmium, whereas some of the other mutants also responded to elevated concentrations of copper and zinc ions. One of the mutants, strain C8, also showed increased gene expression in the presence of the solvent ethanol, but otherwise no overlap between cadmium-induced gene expression and general stress response was detected. Molecular analysis of the corresponding genetic loci was performed using arbitrary polymerase chain reaction (PCR), DNA sequencing and comparison of the deduced protein products with sequences deposited in genetic databases. Some of the genetic loci targeted by the transposon did not show any similarities to any known genes; thus, they may represent 'novel' loci. The hypothesis that genes that are differentially expressed in the presence of heavy metals play a role in metal tolerance was verified for one of the mutants. This mutant, strain C11, was hypersensitive to cadmium and zinc ions. In mutant C11, the transposon had inserted into a genetic region displaying similarity to genes encoding the sensor/regulator protein pairs of two-component systems that regulate gene expression in metal-resistant bacteria, including czcRS of Ralstonia eutropha, czrRS of Pseudomonas aeruginosa and copRS of Pseudomonas syringae. Although the P. fluorescens strain used in this study had not been isolated from a metal-rich environment, it nevertheless contained at least one genetic region enabling it to cope with elevated concentrations of heavy metals.     

Thermophilic mutants of Pseudomonas fluorescens

Summary A series of heat tolerant mutants of Pseudomonas fluorescens were obtained which can grow at temperatures up to 54°C, in contrast to a maximum growth temperature of 37°C for the wild type. The minimum temperatures allowing growth of the mutant strains increased to the same extent as their maximum temperatures. Antibiotic sensitivity patterns suggested the mutants had altered ribosomes, but the purified mutant ribosomes showed no significant increase in thermostability. The virulence of the wild and mutant strains for mice correlated with their relative abilities to grow at the mouse body temperature of approximately 37°C.