Influence of water activity on the ice-nucleating activity of Pseudomonas syringae

Pseudomonas syringae is known as a biological ice-nucleating agent. The bacterium has the unusual property of increasing the temperature at which water freezes by a few degrees. However, the ice-nucleating activity (INA) always remains lower for in vitro cultivated cells, than for cells grown in planta. We examined the effects of the hydrophobic environment and of water availability, on the in vitro growth and INA of P. syringae. The hydrophobic environment was modified by addition of fatty acids, vegetable oils or silicone oil to the culture medium. Addition of olive oil (1%), or traces of silicone oil in the culture medium had a positive effect upon the expression of INA. Variations in water activity from 0.990 to 0.988 by addition of sugar beet fibres or sodium chloride in the culture medium were followed by an increase in INA. This study suggested that control of the medium’s water activity must be considered as an important parameter for optimization of INA in P. syringae. Received 16 June 1998/ Accepted in revised form 02 September 1999     

Identification of a Compound in Chamaecyparis taiwanensis Inhibiting the Ice-nucleating Activity of Pseudomonas fluorescens KUIN-1

Inactivation of the ice-nucleating activity of Pseudomonas fluorescens KUIN-1 by compounds in the leaves from coniferous trees were investigated, and the inactivated material was identified. Intact cells of the strain KUIN-1 and the acetone or methanol extracts of leaves of various coniferous trees were allowed to react for 30 min at 18°C. Antinucleation compounds were obtained from Chamaecyparis taiwanensis. When the acetone extract from the leaves of coniferous trees was added to the cell suspension (about 10⁶ cells/ml) in 50 mM potassium phosphate buffer (pH 7.0), the ice nucleating temperature, T₅₀, was significantly decreased (T₅₀<-5°C). This inhibitor was isolated by using TLC, then identified as hinokitiol based on UV-VIS, IR, and mass spectral data. When intact cells of the strain KUIN-1 were incubated with hinokitiol, limonene, and α-pinene of the principal constituent of the leaves of coniferous trees in 50 mM potassium phosphate buffer (pH 7.0), the ice-nucleating activity decreased, but not in α-terpinene. Furthermore, the ice-nucleating activities from other ice-nucleating bacteria also decreased in the presence of hinokitiol. This inhibition was proportional to the concentration of hinokitinol. The pH and thermal stabilities of the ice-nucleating activity of the cells were changed by the addition of hinokitiol (10 mM).     

丁香假单胞菌冰核基因inaQ变速箱启动子结构与活性分析

序列比对与结构预测显示丁香假单胞菌(Pseudomonas syringae)野生型菌株MB03的冰核基因inaQ启动子为一种在细菌中罕见的变速箱型启动子。通过克隆长度为522bp的inaQ基因启动子区(P522)并与绿色荧光蛋白基因gfp构建融合基因P522gfp后,在恶臭假单胞菌AB92019菌株中进行表达分析。结果表明,包含结构模块A-Box和B-Box的P522在该菌株中具有启动子活性,且在寡营养条件和较低温度下具有更高的活性,是一种可调控启动子。     

Effects of aqueous ozone on Pseudomonas syringae viability and ice nucleating activity

This study reports the impact of different ozone treatments on a Pseudomonas syringae strain known for its ice nucleation activity (INA). Ozone is a very powerful germicidal agent used for water treatment. The effect of ozone on viability and on cultivability of P. syringae was determined by flow cytometry analysis and by plate counting respectively. The impact of ozone on the outer membrane using the INA as marker was investigated by the drop freezing technique.The destruction curve followed a shoulder pattern with a slight reduction in population with CT values between 0 and 8 min. For an initial population of 9.3 log CFU mL^?1, the cultivability was lost starting at 14 min and a loss of viability was observed after 16 min of ozone treatment at 0.45 mg L^?1. Microscopic observations at this point revealed whole but aggregated bacilli. INA decreased after 8 min of ozone treatment but did not disappear. This decrease could be due to the progressive disruption of ice nucleating sites in the outer membrane. It was however partially restored after long storage at 4 °C of dead cells treated for 16 min.     

Properties of a Novel Extracellular Cell-free Ice Nuclei from Ice-nucleating Pseudomonas antarctica IN-74

Some ice-nucleating bacterial strains, including Pantoea ananatis (Erwinia uredovora), Pseudomonas fluorescens, and Pseudomonas syringae isolates, were examined for the ability to shed ice nuclei into the growth medium. A novel ice-nucleating bacterium, Pseudomonas antarctica IN-74, was isolated from Ross Island, Antarctica. Cell-free ice nuclei from P. antarctica IN-74 were different from the conventional cell-free ice nuclei and showed a unique characterization. Cell-free ice nuclei were purified by centrifugation, filtration (0.45 μm), ultrafiltration, and gel filtration. In an ice-nucleating medium in 1 liter of cell culture, maximum growth was obtained with the production of 1.9 mg of cell-free ice nuclei. Ice nucleation activity in these cell-free ice nuclei preparations was extremely sensitive to pH. It was demonstrated that the components of cell-free ice nuclei were protein (33%), saccharide (12%), and lipid (55%), indicating that cell-free ice nuclei were lipoglycoproteins. Also, carbohydrate and lipid stains showed that cell-free ice nuclei contained both carbohydrate and lipid moieties.     

Studies on the cytoplasmic protein tyrosine kinase activity of the Antarctic psychrotrophic bacterium Pseudomonas syringae

The Antarctic psychrotrophic bacterium Pseudomonas syringae contains a 66-kDa cytoplasmic protein which was found to by phosphorylated on a tyrosine residue [Ray, M.K. et al. (1994) FEMS Microbiol. Lett. 122, pp. 49-54]. To investigate the nature of the cytoplasmic protein tyrosine kinase and its role in the bacterial physiology, we carried out some biochemical studies of the enzyme in vitro in the presence of exogenous peptide substrates and expression studies in vivo at low and high temperature during various phases of growth. The results suggest that the protein tyrosine kinase associated with the cytoplasmic fraction of the bacterium has certain similarities and dissimilarities with the known eukaryotic tyrosine kinases. The protein tyrosine kinase could phosphorylate exogenous substrate corresponding to the N-terminal peptide of p34cdc2 kinase but could not do so on poly(Glu:Tyr). The enzyme could not be inhibited by genistein, staurosporine and dimethyl aminopurine, but could be inhibited by piceatannol which is a known competitive inhibitor of the peptide binding site of mammalian protein tyrosine kinases. The enzyme activity in the cytoplasm is uniquely inhibited by sodium orthovanadate (IC50 = 20 microM) which is a known protein tyrosine phosphatase inhibitor. The expression studies show that the enzyme is produced more at a higher temperature (22 degrees C) of growth than at lower temperature (4 degrees C) and during the stationary phase of growth of P. syringae.     

Identification and Characterization of Pseudomonas syringae pv. syringae,a Causative Bacterium of Apple Canker in Korea

In the present investigation, bacterial isolates from infected apple trees causing apple canker during winter were studied in the northern Gyeongbuk Province, Korea. The pathogen was identified as Pseudomonas syringae pv. syringae (Pss) through various physiological and biochemical characterization assays such as BIOLOG, gas chromatography of fatty acid methyl esters, and 16S rRNA. Bioassays for the production of phytotoxins were positive for syringopeptin and syringomycin against Bacillus megaterium and Geotrichum candidum, respectively. The polymerase chain reaction (PCR) method enabled the detection of toxin-producing genes, syrB1, and sypB in Pss. The differentiation of strains was performed using LOPAT and GATTa tests. Pss further exhibited ice nucleation activity (INA) at a temperature of −0.7°C, indicating an INA⁺ bacterium. The ice-nucleating temperature was −4.7°C for a non-treated control (sterilized distilled water), whereas it was −9.6°C for an INA⁻ bacterium Escherichia coli TOP10. These methods detected pathogenic strains from apple orchards. Pss might exist in an apple tree during ice injury, and it secretes a toxin that makes leaves yellow and cause canker symptoms. Until now, Korea has not developed antibiotics targeting Pss. Therefore, it is necessary to develop effective disease control to combat Pss in apple orchards. Pathogenicity test on apple leaves and stems showed canker symptoms. The pathogenic bacterium was re-isolated from symptomatic plant tissue and confirmed as original isolates by 16S rRNA. Repetitive element sequence-based PCR and enterobacterial repetitive intergenic consensus PCR primers revealed different genetic profiles within P. syringae pathovars. High antibiotic susceptibility results showed the misreading of mRNA caused by streptomycin and oxytetracycline.     

Non contiguous-finished genome sequence of Pseudomonas syringae pathovar syringae strain B64 isolated from wheat

The Gram-negative gammaproteobacterium Pseudomonas syringae is one of the most wide-spread plant pathogens and has been repeatedly reported to cause significant damage to crop plantations. Research on this pathogen is very intensive, but most of it is done on isolates that are pathogenic to Arabidopsis, tomato, and bean. Here, we announce a high-quality draft genome sequence of Pseudomonas syringae pv. syringae B64 which is the first published genome of a P. syringae strain isolated from wheat up to date. The genome sequence will assist in gaining insights into basic virulence mechanisms of this pathogen which has a relatively small complement of type III effectors.     

丁香假单胞菌环式脂肽毒素的生理和分子生物学研究

综合评述了近10年来在丁香假单胞菌脂肽毒素生理和分子生物学研究上的发现。这些毒素依肽部AA数目可分两组。丁香假单胞霉素组(syringomycuns)已报告4个成员,肽部有9个AA;丁香假单胞肽毒素组有2个成员,肽部分别有22个和25个AA。肽部C端羧基与分子内羟基氨基酸残基(AA)的羟基酯化闭合成环,再由羟基脂肪酸酰化。两组毒素都诱导植物电解质渗漏、人和动物红血球溶解,其机制在于在细胞膜上形成二价阳离子可通过的寡体通道。对酵母菌的抑制作用受固醇的种类影响,以胆固醇的保护作用最强。丁香假单胞霉素的合成涉及一个多酶系统,有些负责肽合成,有些负责运输或调节,除受内源调节蛋白调节外,也受外源信号分子调节,尤其是受植物酚糖苷诱导。这些毒素具有抗真菌活性,对人和动物的一些病原霉菌有明显效果,在试验剂量无副作用,在医药上应用的前景良好。     

The influence of natural mineral water on aquaporin water permeability and human natural killer cell activity

Aquaporins are the intrinsic membrane proteins functioning as water channel to transport water and/or mineral nutrients across the biological membrane systems. In this research, we aimed to clarify if the selected mineral water can affect aquaporin functions in vitro and the assumption of the mineral water can modify aquaporin expression and activate natural killer cell activity in human body. First, we expressed six human and eight plant aquaporin genes in oocytes and compared the effect of different kinds of natural mineral water on aquaporin activity. The oocyte assay data show that Hita tenryosui water could promote water permeability of almost all human and plant aquaporins in varying degrees, and freeze-dry and organic solvent extraction could reduce AQP2 activity but pH change and boiling could not. Second, each volunteer in two groups (10 in one group) received an oral Hita tenryosui or tap water load of 1000 ml/day for total four weeks. We found that these two kinds of water did not directly affect the relative expression levels of AQP1 and AQP9 in the blood cells, but intriguingly, the natural killer cell activities of the volunteers drinking Hita tenryosui water were significantly improved, suggesting that Hita tenryosui water has obvious health function, which opens a new and interesting field of investigation related to the link between mineral water consumption and human health and the therapies for some chronic diseases.     

发酵麸皮多糖酶辅助提取工艺优化及其生物活性研究

通过响应面法优化提取发酵麸皮多糖的工艺,并评价其体外益生和抗氧化活性。以发酵麸皮多糖的得率为响应值,采用纤维素酶酶解与水浴浸提相结合的方法提取发酵麸皮多糖,以纤维素酶添加量、料液比、水浴浸提温度、水浴浸提时间为试验因素建立数学模型,筛选最佳提取工艺条件。通过测定还原力、DPPH和·OH自由基的清除能力对比发酵和未发酵麸皮多糖的体外抗氧化活性,并通过测定嗜酸乳杆菌、植物乳杆菌、两歧双歧杆菌的生长对比发酵和未发酵麸皮多糖的体外益生活性。结果表明,发酵麸皮多糖最佳提取工艺为:料液比1∶16(w/v),酶添加量1 000 U/g,水浴浸提温度90℃,水浴浸提时间60 min,在此条件下发酵麸皮多糖的得率实测值为73. 35%。发酵麸皮多糖具有较强的DPPH和·OH自由基的清除能力,可促进嗜酸乳杆菌、植物乳杆菌和两歧双歧杆菌的生长。     

Characterization of a nonspecific phosphopantetheinyl transferase from Pseudomonas syringae pv. syringae FF5

The 4'-phosphopantetheinyl transferases (PPTases) catalyze the transfer of a 4'-phosphopantetheine moiety from coenzyme A to phosphopantetheine-dependent carrier proteins. The carrier proteins (CPs) are required for the biosynthesis of peptides synthesized by nonribosomal peptide synthases and the biosynthesis of fatty acids and polyketides. A single PPTase (PcpS) is present in the pathogenic bacterium Pseudomonas aeruginosa. Several pathovars of Pseudomonas syringae produce the chlorosis-inducing phytotoxin coronatine. Structural genes for coronatine biosynthesis include two ACPs, two ACP domains, and one peptidyl carrier protein (PCP) domain. To gain insight into factors affecting coronatine biosynthesis, the PPTase of P. syringae pv. syringae FF5 has been investigated. A single PPTase gene (pspT) was amplified from this organism by PCR. The translation product PspT exhibited 62% identity to PcpS as well as higher levels of identity to other, uncharacterized Pseudomonad PPTases. PspT was overproduced in soluble form in Escherichia coli and its enzymatic properties were compared with those of PcpS. PspT exhibited broad substrate specificity, and it displayed the highest activity with a PCP domain. In contrast, the most efficient substrates for PcpS are CPs from primary metabolism. These results indicate phosphopantetheinyl transferases from different Pseudomonas sp. may vary significantly in their enzymatic properties.     

Pseudomycins, a family of novel peptides from Pseudomonas syringae possessing broad-spectrum antifungal activity.

A family of peptide antimycotics, termed pseudomycins, has been isolated from liquid cultures of Pseudomonas syringae, a plant-associated bacterium. These compounds were purified using Amberlite XAD-2 and reverse-phase liquid chromatography. Pseudomycin A, the predominant peptide in a family of four, showed selective phytotoxicity, and had impressive activity against the human pathogen Candida albicans. Amino acid, mass spectroscopic, and comparative electrophoretic and chromatographic analyses revealed that the pseudomycins are different from previously described antimycotics from P. syringae, including syringomycin, syringotoxin and syringostatins. Pseudomycins A-C contain hydroxyaspartic acid, aspartic acid, serine, arginine, lysine and diaminobutyric acid. The molecular masses of pseudomycins A-C, as determined by plasma desorption mass spectrometry, are 1224, 1208 and 1252 Da, respectively. Pseudomycin D, on the other hand, has a molecular mass of 2401 Da and is more complex than pseudomycins A-C.     

Cytokinin activity of zeatin allylic phosphate, a natural compound

Zeatin allylic phosphate (ZAP) retarded chlorophyll loss in the barleyleaf senescence assay at a concentration 20 times higher than for6-benzyladenine (BA): the effective concentrations for ZAP and BA were 10 and 0.5 , respectively. Sodium molybdate,an inhibitor of phosphatases, decreased the ZAP effective concentration to 0.5 without affecting leaf senescence andtrans-zeatin activity in the control. This demonstrates theimportance of the phosphate group for ZAP activity or its penetration into leafcells. ZAP up-regulated the protein kinase activity of the barley leaf chromatinwith concentration dependence similar to that oftrans-zeatin. Conversely, ZAP was 1000 times less activethan trans-zeatin in the competition with anti-idiotypeantibodies (raised against antibody to zeatin) for binding with atrans-zeatin-binding site oftrans-zeatin-binding protein ZBP67 isolated from barleyleaves. In contrast to trans-zeatin, ZAP did not activateRNA synthesis in the presence of ZBP in the in vitro systemcontaining chromatin and RNA polymerase I isolated from barley leaves. Insummary, data presented show that ZAP possesses cytokinin activity asdemonstrated by the retardation of barley leaf senescence, but moleculartarget(s) for ZAP in barley leaf cells differs, at least partially, from thesefor trans-zeatin. It seems possible that the cytokininactivity of ZAP results from its hydrolysis while producing zeatin.     

Enhanced phytase production from Achromobacter sp. PB‐01 using wheat bran as substrate: Prospective application for animal feed

This article deals with the optimization of the various parameters for production of phytase using Achromobacter sp. PB‐01 in submerged fermentation (SmF). A semisynthetic medium containing ingredients of phytase screening media (PSM) supplemented with 2% (w/v) sucrose, 1% (w/v) peptone, and 10% (w/v) wheat bran was found to be the best production medium among the various combinations tried. Among various surfactants added to SmF, Triton X‐100 (0.1%) exhibited a 16% increase in phytase activity. An overall 11.2 fold enhancement in enzyme activity (0.79 U/mL→8.84 U/mL) was attained when SmF was carried out using 0.5% (v/v) inoculum of a 15 h old culture of Achromobacter sp. PB‐01 at an initial pH of 5.5, temperature 30°C and allowed to grow for 48 h. Presence of accessory hydrolytic enzymes in the crude extract further added value as feed additive by mediating efficient degradation of non‐starch polysaccharides (NSP). In addition, we also investigated the efficacy of phytase on different agro‐industrial residues using in vitro experiments that simulated the conditions of the digestive tract. Results indicate that phytase from our source hydrolyze phytate efficiently with the concomitant liberation of inorganic phosphate, protein, reducing sugar, and calcium. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Contribution of Nitrate Assimilation to the Fitness of Pseudomonas syringae pv. syringae B728a on Plants

The ability of Pseudomonas syringae pv. syringae to use nitrate as a nitrogen source in culture and on leaves was assessed. Substantial amounts of leaf surface nitrate were detected directly and by use of a bioreporter of nitrate on bean plants grown with a variety of nitrogen sources. While a nitrate reductase mutant, P. syringae ΔnasB, exhibited greatly reduced growth in culture with nitrate as the sole nitrogen source, it exhibited population sizes similar to those of the wild-type strain on leaves. However, the growth of the ΔnasB mutant was much less than that of the wild-type strain when cultured in bean leaf washings supplemented with glucose, suggesting that P. syringae experiences primarily carbon-limited and only secondarily nitrogen-limited growth on bean leaves. Only a small proportion of the cells of a green fluorescent protein (GFP)-based P. syringae nitrate reductase bioreporter, LK2(pOTNas4), exhibited fluorescence on leaves. This suggests that only a subset of cells experience high nitrate levels or that nitrate assimilation is repressed by the presence of ammonium or other nitrogenous compounds in many leaf locations. While only a subpopulation of P. syringae consumes nitrate at a given time on the leaves, the ability of those cells to consume this resource would be strongly beneficial to those cells, especially in environments in which nitrate is the most abundant form of nitrogen.     

Erwinia herbicola冰核活性蛋白的分离、电泳分析鉴定

对Erwinia herbicola(A25)菌株的冰核活性蛋白的分离纯化及电泳进行研究。主要方法①按双温培养的方法,获得了较高的生物量积累和强冰核活性的诱导表达。②实验采用渗透压冲击法破碎细菌细胞,破碎率达98．67％。③通过差速离心法获取不同冰核活性蛋白组分,测定各组分的冰核活性和SDS－PAGE电泳图谱分析。建立了冰核活性因子的高冰核活性与离心力之间的关系;利用SDS－PAGE还建立了具冰核活性蛋白的分子量的大小与冰核活性蛋白组分之间的关系。证实了具高冰核活性蛋白质最小结构单位约为26．0kD。     

The influence of moisture on the suppression of Pseudomonas syringae by Aureobasidium pullulans on an artificial leaf surface

Abstract Isolates of Aureobasidium pullulans which produce antibacterial metabolites were compared with non-antibiotic-producing isolates of Tremella foliacea and Trichosporon beigelii for their ability to inhibit growth of Pseudomonas syringae pv phaseolicola . The bacteria and fungi were co-inoculated on an artificial leaf surface under conditions of low and high water availability. Under conditions of excess moisture, antibiotic production gave no advantage to the Aureobasidium isolates, and all the yeasts were equally antagonistic. Under drier conditions the Aureobasidium isolates reduced the population of P. syringae by 96–99%, a result which was significantly different from that which occurred when the bacteria were co-inoculated with the other yeasts under these conditions. An antibacterial compound, similar to that produced in liquid culture by Aureobasidium , was detected in washings from the artificial leaf surface following growth of this species.     

Crystal structure of uroporphyrinogen III synthase from Pseudomonas syringae pv. tomato DC3000

Uroporphyrinogen III synthase (U3S) is one of the key enzymes in the biosynthesis of tetrapyrrole compounds. It catalyzes the cyclization of the linear hydroxymethylbilane (HMB) to uroporphyrinogen III (uro’gen III). We have determined the crystal structure of U3S from Pseudomonas syringae pv. tomato DC3000 (psU3S) at 2.5 Å resolution by the single wavelength anomalous dispersion (SAD) method. Each psU3S molecule consists of two domains interlinked by a two-stranded antiparallel β-sheet. The conformation of psU3S is different from its homologous proteins because of the flexibility of the linker between the two domains, which might be related to this enzyme’s catalytic properties. Based on mutation and activity analysis, a key residue, Arg219, was found to be important for the catalytic activity of psU3S. Mutation of Arg219 to Ala caused a decrease in enzymatic activity to about 25% that of the wild type enzyme. Our results provide the structural basis and biochemical evidence to further elucidate the catalytic mechanism of U3S.