49 resultados para Pseudomonas Putida


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采用PCR-DGGE方法,对我国东北地区长期石油和重金属污染农田土壤中的假单胞菌多样性及其种群结构进行研究.结果表明:石油污染区土壤中假单胞菌多样性指数显著高于重金属污染区;石油污染区旱田土壤假单胞菌多样性接近于对照清洁土壤,但低于相似污染程度的石油污染区水田土壤,表明污染物类型与耕作管理方式是影响土壤中假单胞菌多样性的主要因素.经16SrRNA的V6/V7区测序,Pseudomonas mendocina、P.stutzeri和P.aerugi-nosa是该石油和重金属污染区土壤中的优势类群,说明在长期污染胁迫下,这3种假单胞菌分别得到了不同程度的富集,推测与石油烃的自然降解及假单胞菌的重金属抗性有关.

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为获得更为丰富的石油降解微生物资源,从沈抚污灌区石油污染土壤和实验室高浓度柴油胁迫土壤中筛选出了4株高效石油烃降解菌SF-422、SF-428、SF-433和SYS-1.这4株菌总石油烃(Total petroleum hydrocarbon/TPH)生物降解率为67.4%~73.6%.经过16项生理生化特性实验和16S rDNA序列分析鉴定,SF-433,SF-428,SF-422和SYS-1分别为蜡状芽孢杆菌(Bacillus cereus),木糖氧化无色杆菌(Achromobacter xylosoxidans),施氏假单胞菌(Pseudomonas stutzeri)和洋葱伯克霍尔德氏菌(Burkholderia cepacia).纯烃降解定性实验表明所筛选出的4株高效降解菌均能够利用正十六烷、苯、菲和环己烷为唯一碳源生长,其中菌株SF-428和SYS-1显示了对芳烃及环烷烃较强的利用能力.

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Problems with tin and copper antifouling compounds have highlighted the need to develop new environmentally friendly antifouling coatings. Bacteria isolated from living surfaces in the marine environment are a promising source of natural antifouling compounds. Four isolates were used to produce extracts that were formulated into ten waterbased paints. All but one of the paints showed activity against a test panel of fouling bacteria. Five of the paints were further tested for their ability to inhibit the settlement of barnacle larvae, Balanus amphitrite, and algal spores of Ulva lactuca, and for their ability to inhibit the growth of U. lactuca. Two paints caused a significant decrease in the number of settled barnacles. One paint containing extract of Pseudomonas sp. strain NUDMB50-11, showed excellent activity in all assays. The antifouling chemicals responsible for the activity of the extract were isolated, using bioassay guided fractionation, and their chemical structures determined.

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Blend films of poly(epsilon-caprolactone) (PCL) and poly(DL-lactide) (PDLLA) with 0.5 weight fraction of PCL were prepared by means of solution casting and their degradation behavior was studied in phosphate buffer solution containing Pseudomonas (PS) lipase. Enzymatic degradation of the blend films occurred continuously within the first 6 days and finally stopped when the film weight loss reached 50%, showing that only PCL in the blends degraded under the action of PS lipase in the buffer solution. These results indicate the selectivity of PS lipase on the promotion of degradation for PCL and PDLLA. The thermal properties and morphology of the blend films were investigated by differential scanning calorimetry, wide-angle X-ray diffraction and scanning electron microscopy (SEM). The morphology resulting from aggregate structures of PCL in the blends was destroyed in the enzymatic degradation process, as observed by SEM. These results confirm again the enzymatic degradation of PCL in the blends in the presence of PS lipase. (C) 1999 Published by Elsevier Science Ltd. All rights reserved.

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A successful micronization of water-insoluble poly(epsilon-caprolactone) (PCL) into narrowly distributed nanoparticles stable in water has not only enabled us to study the enzymatic biodegradation of PCL in water at 25 degrees C by a combination of static and dynamic laser light scattering (LLS), but also to shorten the biodegradation time by a factor of more than 10(3) compared with using a thin PCL film, i.e. a 1 week conventional experiment becomes a 4 min one. The time-average scattering intensity decreased linearly. It was interesting to find that the decrease of the scattering intensity was not accompanied by a decrease of the average size of the PCL nanoparticles, indicating that the enzyme, Lipase Pseudomonas (PS), ''eats'' the PCL nanoparticles one-by-one, so that the biodegradation rate is determined mainly by the: enzyme concentration. Moreover, we found that using anionic sodium lauryl sulphate instead of cationic hexadecyltrimethylammonium bromide as surfactant in the micronization can prevent the biodegradation, suggesting that the biodegradation involves two essential steps: the adsorption of slightly negatively charged Lipase PS onto the PCL nanoparticles and the interaction between Lipase PS and PCL. (C) 1999 Elsevier Science Ltd. All rights reserved.

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A novel combination of laser light scattering (LLS) and the micronization of a water-insoluble polymer into narrowly distributed nanoparticles stable in water has provided not only an accurate, reliable and microscopic method to study polymer biodegradation, but also a novel and fast way to evaluate the biodegradability of a given polymer. Using poly(epsilon-caprolactone) (PCL) as a typical example, we have shown that its biodegradation time can be shortened by a factor of more than 10(3) times in comparison with the time required to biodegrade a thin film (10 x 10 x 0.1 mm(3)). Moreover, the biodegradation kinetics can be in-situ monitored in terms of the decrease of the time-average scattering intensity and the particle number. A comparison of static and dynamic LLS results revealed that the enzyme, Lipase Pseudomonas, ''eats'' the PCL nanoparticles in an one-by-one manner and the enzymatic biodegradation of PCL follows a zero-order kinetics. (C) 1998 Elsevier Science Ltd. All rights reserved.

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The enzymatic degradation of poly(epsilon-caprolactone) (PCL) films in phosphate buffer solution containing lipases has been studied by DSC, WAXD and SEM. Three lipases, pseudomonas lipase (PS), porcine pancreatic lipase (PP), and candida cylindracea lipase (AY), were used. The results showed that the degradation of PCL films in phosphate buffer solution containing PP or AY was very slow: no weight loss could be found within 1 week. However, PCL film could degrade rapidly and completely within 4 days in phosphate buffer solution containing PS lipase. (C) 1997 Elsevier Science Limited.

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Edwardsiella tarda is an important Gram-negative enteric pathogen affecting both animals and humans. It possesses a type III secretion system (T3SS) essential for pathogenesis. EseB, EseC and EseD have been shown to form a translocon complex after secretion, while EscC functions as a T3SS chaperone for EseB and EseD. In this paper we identify EscA, a protein required for accumulation and proper secretion of another translocon component, EseC. The escA gene is located upstream of eseC and the EscA protein has the characteristics of T3SS chaperones. Cell fractionation experiments indicated that EscA is located in the cytoplasm and on the cytoplasmic membrane. Mutation with in-frame deletion of escA greatly decreased the secretion of EseC, while complementation of escA restored the wild-type secretion phenotype. The stabilization and accumulation of EseC in the cytoplasm were also affected in the absence of EscA. Mutation of escA did not affect the transcription of eseC but reduced the accumulation level of EseC as measured by using an EseC-LacZ fusion protein in Ed. tarda. Co-purification and co-immunoprecipitation studies demonstrated a specific interaction between EscA and EseC. Further analysis showed that residues 31-137 of EseC are required for EseC-EscA interaction, Mutation of EseC residues 31-137 reduced the secretion and accumulation of EseC in Ed. tarda. Finally, infection experiments showed that mutations of EscA and residues 31-137 of EseC increased the LD50 by approximately 10-fold in blue gourami fish. These results indicated that EscA functions as a specific chaperone for EseC and contributes to the virulence of Ed. tarda.

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Edwardsiella tarda is a gram-negative pathogen with a broad host range that includes humans, animals, and fish. Recent studies have shown that the LuxS/autoinducer type 2 (AI-2) quorum sensing system is involved in the virulence of E. tarda. In the present study, it was found that the E. tarda LuxS mutants bearing deletions of the catalytic site (C site) and the tyrosine kinase phosphorylation site, respectively, are functionally inactive and that these dysfunctional mutants can interfere with the activity of the wild-type LuxS. Two small peptides, 5411 and 5906, which share sequence identities with the C site of LuxS, were identified. 5411 and 5906 proved to be inhibitors of AI-2 activity and could vitiate the infectivity of the pathogenic E. tarda strain TX1. The inhibitory effect of 5411 and 5906 on AI-2 activity is exerted on LuxS, with which these peptides specifically interact. The expression of 5411 and 5906 in TX1 has multiple effects (altering biofilm production and the expression of certain virulence-associated genes), which are similar to those caused by interruption of luxS expression. Further study found that it is very likely that 5411 and 5906 can be released from the strains expressing them and, should TX1 be in the vicinity, captured by TX1. Based on this observation, a constitutive 5411 producer (Pseudomonas sp. strain FP3/pT5411) was constructed in the form of a fish commensal isolate that expresses 5411 from a plasmid source. The presence of FP3/pT5411 in fish attenuates the virulence of TX1. Finally, it was demonstrated that fish expressing 5411 directly from tissues exhibit enhanced resistance against TX1 infection.

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Edwardsiella tarda is a gram-negative pathogen with a broad host range that includes humans, animals, and fish. Recent studies have shown that the LuxS/autoinducer type 2 (AI-2) quorum sensing system is involved in the virulence of E. tarda. In the present study, it was found that the E. tarda LuxS mutants bearing deletions of the catalytic site (C site) and the tyrosine kinase phosphorylation site, respectively, are functionally inactive and that these dysfunctional mutants can interfere with the activity of the wild-type LuxS. Two small peptides, 5411 and 5906, which share sequence identities with the C site of LuxS, were identified. 5411 and 5906 proved to be inhibitors of AI-2 activity and could vitiate the infectivity of the pathogenic E. tarda strain TX1. The inhibitory effect of 5411 and 5906 on AI-2 activity is exerted on LuxS, with which these peptides specifically interact. The expression of 5411 and 5906 in TX1 has multiple effects (altering biofilm production and the expression of certain virulence-associated genes), which are similar to those caused by interruption of luxS expression. Further study found that it is very likely that 5411 and 5906 can be released from the strains expressing them and, should TX1 be in the vicinity, captured by TX1. Based on this observation, a constitutive 5411 producer (Pseudomonas sp. strain FP3/pT5411) was constructed in the form of a fish commensal isolate that expresses 5411 from a plasmid source. The presence of FP3/pT5411 in fish attenuates the virulence of TX1. Finally, it was demonstrated that fish expressing 5411 directly from tissues exhibit enhanced resistance against TX1 infection.

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C-type lectins are a superfamily of carbohydrate-recognition proteins which play crucial roles in the innate immunity. In this study, the gene of a C-type lectin with multiple carbohydrate-recognition domains (CRDs) from scallop Chlamys farreri (designated as Cflec-3) was cloned by rapid amplification of cDNA ends (RACE) approach based on expression sequence tag (EST) analysis. The full-length cDNA of Cflec-3 was of 2256 bp. The open reading frame encoded a polypeptide of 516 amino acids, including a signal sequence and three CRDs. The deduced amino acid sequence of Cflec-3 showed high similarity to members of C-type lectin superfamily. By fluorescent quantitative real-time PCR, the Cflec-3 mRNA was mainly detected in hepatopancreas, adductor, mantle, and marginally in gill, gonad and hemocytes of healthy scallops. After scallops were challenged by Listonella anguillarum, the mRNA level of Cflec-3 in hemocytes was up-regulated and was significantly higher than that of blank at 8 h and 12 h post-challenge. The function of Cflec-3 was investigated by recombination and expression of the cDNA fragment encoding its mature peptide in Escherichia coli BL21 (DE3)-pLysS. The recombined Cflec-3 (rCflec-3) agglutinated Gram-negative bacteria Pseudomonas stutzeri. The agglutinating activity was calcium-dependent and could be inhibited by D-mannose. These results collectively suggested that Cflec-3 was involved in the immune response against microbe infection and contributed to nonself-recognition and clearance of bacterial pathogens in scallop. (C) 2009 Elsevier Ltd. All rights reserved.

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Scanning electron microscopy of the surfaces of the seaweeds Laminaria japonica, haploid Porphyra yezoensis, Ulva pertusa and the diploid conchocelis of P. yezoensis and P. haitanensis revealed Vibrio and Micrococcus to be abundant on the surfaces of U. pertusa and P. yezoensis. Vibrio, Flavobacterium, Pseudomonas, Staphylococcus, Bacillus, Corynebacterium and other genera were isolated from the surfaces of L. japonica.

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A gene encoding a chitosanase (mschito) was cloned from Microbacterium, sp. OU01. The ORF consists of 801 bp which encoded a polypeptide of 266 amino acid residues. The deduced amino acid sequence shows 98% identity to that of the chitosanase reported in Pseudomonas sp. A-01. In addition, the fusion protein containing MSCHITO was expressed in E. coli and purified using Ni-NTA affinity chromatography. The purified rMSCHITO protein degraded the chitosan (the degree of deacetylation of 99%) and produced a mixture of chitooligosaccharides. The MSCHITO is thus an endo-chitosanase.

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Protease-producing bacteria are known to play an important role in degrading sedimentary particular organic nitrogen, and yet, their diversity and extracellular proteases remain largely unknown. In this paper, the diversity of the cultivable protease-producing bacteria and their extracellular proteases in the sediments of the South China Sea was investigated. The richness of the cultivable protease-producing bacteria reached 10(6) cells/g in all sediment samples. Analysis of the 16S rRNA gene sequences revealed that the predominant cultivated protease-producing bacteria are Gammaproteobacteria affiliated with the genera Pseudoalteromonas, Alteromonas, Marinobacter, Idiomarina, Halomonas, Vibrio, Shewanella, Pseudomonas, and Rheinheimera, with Alteromonas (34.6%) and Pseudoalteromonas (28.2%) as the predominant groups. Inhibitor analysis showed that nearly all the extracellular proteases from the bacteria are serine proteases or metalloproteases. Moreover, these proteases have different hydrolytic ability to different proteins, reflecting they may belong to different kinds of serine proteases or metalloproteases. To our knowledge, this study represents the first report of the diversity of bacterial proteases in deep-sea sediments.

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本论文报道从海洋中分离到的一株聚磷菌的分离、鉴定、在系统发育中的地位、除磷特性、菌体内多磷酸盐颗粒的研究、D-海因酶和核苷二磷酸激酶基因的克隆及序列分析,为海水系统的生物除磷提供部分基础资料。 从黄海海域分离到聚磷菌Halomonas sp. YSR-3,菌体呈杆状,大小为3.5 μm×1 μm,革兰氏阴性,好氧生长,能运动。透射电镜观察发现,菌体内有致密颗粒。经DAPI染色确定该致密颗粒是多磷酸盐,亦可称为异染粒、迂回体。16S rDNA鉴定结果表明,YSR-3与Halomonas属中的marine bacterium B5-7有较高的同源性,相似值99%。YSR-3的生理生化特性:对氯霉素和卡那霉素敏感;淀粉水解呈阳性;反硝化和几丁质降解呈阴性;能将葡萄糖作为唯一碳源和能源。 对YSR-3的培养条件进行优化。以海水2216培养基、24 ℃、180 rpm、pH 6.5的条件培养,更利于菌体生长和菌体内多磷酸盐的形成。 对YSR-3的除磷特性进行研究。无磷培养时,菌体不能生长;用磷酸钾盐作为磷源时,菌体生长较好,形成多磷酸盐的菌体比例较高;较适合YSR-3菌体生长和多磷酸盐形成的磷源是KH2PO4,较适磷浓度为1.5 mmol/L。pH的变化影响菌株的生长、多磷酸盐形成和除磷效果。pH值为5时,菌体的数量几乎不增加,体内多磷酸盐和培养基中磷含量变化不大;pH值为6、7和8时,菌体生长良好,95%以上的菌体内形成多磷酸盐,培养基中磷含量明显下降。YSR-3在不同培养基中除磷量和除磷率不同。在高磷培养基中除磷量为0.7 mmol/L(磷含量由1.84 mmol/L降到1.14 mmol/L),除磷率为37.5%;在低磷培养基中除磷量为0.02 mmol/L(磷含量由0.028 mmol/L降到0.008 mmol/L),除磷率为72.2%。 以海洋聚磷菌Halomonas sp. YSR-3的总DNA为模板,用PCR法扩增D-海因酶基因和核苷二磷酸激酶基因,将扩增片段克隆到pGM-T载体,转化E.coli TOP10菌株,经蓝白斑筛选、菌落PCR得到阳性克隆,测序后对序列进行Blast比对分析。得到的D-海因酶基因序列长度为1510 bp,与Pseudomonas entomophila L48的海因酶基因序列的相似性为77%。翻译后的序列与Pseudomonas fluorescens Pf-5,Marinomonas sp. MED121,Burkholderia vietnamiensis G4的海因酶蛋白序列相似性分别为75%,73%,70%。得到的核苷二磷酸激酶基因序列长度为420bp,翻译后的序列与Loktanella vestfoldensis SKA53,Jannaschia sp. CCS1,Roseobacter sp. CCS2的核苷二磷酸激酶蛋白序列相似性分别为89%,86%,85%。 聚磷菌能将外界环境中的磷吸收到体内,并以多磷酸盐的形式储存。多磷酸盐对于细胞的生存和生长有很重要的作用,但目前对于多磷酸盐的形成过程以及过程调控还不是很清楚。在今后可以通过构建高效表达的重组菌,提高与除磷相关的酶的纯度及活性。同时可以将相关酶的基因进行突变,对基因表达的调控以及酶的代谢以及功能结构等多方面进行基础研究,使聚磷菌在生物除磷中得到广泛应用。