CLONING IN Yersinia enterocolitica OF K99 ANTIGEN GENE FROM Escherichia coli

A recombinant plasmid pFS239 containing the geoe coding for K99 antigen of Escherichia coli and wide-host-range plasmid pKT230 has been cloned in E. coli C600. pFS239 has been transferred to Yersinia enterocolitica strains D29, L15 and L15 (pYV15) through triparental mating. In Y. enterocolitica transconjugants the expression of VW antigens and calcium dependence which represent the propertics associated with the virulence plastmid of Y. enterocolitica remains unchanged.     

Label-free detection of a bacterial pathogen using an immobilized siderophore, deferoxamine

Pathogenic bacteria obtain the iron necessary for survival by releasing an iron chelator, termed a siderophore, and retrieving the iron-siderophore complex via a cell surface siderophore receptor. We have exploited the high affinity of Yersinia enterocolitica for its siderophore, deferoxamine, to develop a rapid method for capture and identification of Yersinia. In this methodology, a deferoxamine-bovine serum albumin conjugate is printed onto a gold-plated chip in a parallel line pattern. After flowing a suspension of Yersinia across the siderophore-derivatized chip, any Yersinia that binds to the chip is detected by dark-field microscopy analysis of the scattered light, followed by Fourier transform analysis of the scattering pattern. Since peak intensities are found to correlate with pathogen concentration, pathogen titers as low as 10(3) cfu/ml can be readily detected. Moreover, immobilized deferoxamine can distinguish Y. enterocolitica, which binds ferrioxamine (deferoxamine-Fe), from Staphylococcus aureus, Mycobacterium smegmatis and Pseudomonas aeruginosa, which don't. Because human pathogens cannot easily mutate their iron retrieval systems without loss of viability, we suggest that few if any mutant Yersinia will emerge that can avoid detection. Together with previous results demonstrating selective capture of Pseudomonas aeruginosa by its immobilized siderophore (pyoverdin), these data suggest that pathogen-specific siderophores may constitute effective and immutable capture ligands for rapid detection and identification of their cognate pathogens.     

Bioinformatics analysis of a non-specific nuclease from Yersinia enterocolitica subsp. palearctica

In this paper, the physical and chemical characteristics, biological structure and function of a non-specific nuclease from Yersinia enterocolitica subsp. palearctica (Y. NSN) found in our group were studied using multiple bioinformatics approaches. The results showed that Y. NSN had 283 amino acids, a weight of 30,692.5 ku and a certain hydrophilic property. Y. NSN had a signal peptide, no transmembrane domains and disulphide bonds. Cleavage site in Y. NSN was between pos. 23 and 24. The prediction result of the secondary structure showed Y. NSN was a coil structure-based protein. The ratio of α-helix, β-folded and random coil were 18.73%, 16.96% and 64.31%, respectively. Active sites were pos. 124, 125, 127, 157, 165 and 169. Mg²⁺ binding site was pos. 157. Substrate binding sites were pos. 124, 125 and 169. The analysis of multisequencing alignment and phylogenetic tree indicated that Y. NSN shared high similarity with the nuclease from Y. enterocolitica subsp. enterocolitica 8081. The enzyme activity results showed that Y. NSN was a nuclease with good thermostability.     

Ferrioxamine B analogues: targeting the FoxA uptake system in the pathogenic Yersinia enterocolitica

A series of ferrioxamine B analogues that target the bacterium Yersinia enterocolitica were prepared. These iron carriers are composed of three hydroxamate-containing monomeric units. Two identical monomers consist of N-hydroxy-3-aminopropionic acid coupled with beta-alanine, and a third unit at the amino terminal is composed of N-hydroxy-3-aminopropionic acid and one of the following amino acids: beta-alanine (1a), phenylalanine (1b), cyclohexylalanine (1c), or glycine (1d). Thermodynamic results for representatives of the analogues have shown a strong destabilization (3-4 orders of magnitude) of the ferric complexes with respect to ferrioxamine B, probably due to shorter spacers and a more strained structure around the metal center. No significant effect of the variations at the N-terminal has been observed on the stability of the ferric complexes. By contrast, using in vivo radioactive uptake experiments, we have found that these modifications have a substantial effect on the mechanism of iron(III) uptake in the pathogenic bacteria Yersinia enterocolitica. Analogues 1a and 1d were utilized by the ferrioxamine B uptake system (FoxA), while 1b and 1c either used different uptake systems or were transported to the microbial cell nonspecifically by diffusion via the cell membrane. Transport via the FoxA system was also confirmed by uptake experiments with the FoxA deficient strain of Yersinia enterocolitica. A fluorescent marker, attached to 1a in a way that did not interfere with its biological activity, provided additional means to monitor the uptake mechanism by fluorescence techniques. Of particular interest is the observation that 1a was utilized by the uptake system of ferrioxamine B in Yersinia enterocolitica (FoxA) but failed to use the ferrioxamine uptake route in Pseudomonas putida. Here, we present a case in which biomimetic siderophore analogues deliberately designed for a particular bacterium can distinguish between related uptake systems of different microorganisms.     

252Cf-plasma desorption mass spectrometry of unmodified lipid A: fragmentation patterns and localization of fatty acids.

The fragmentation patterns of synthetic Escherichia coli-type lipid A in plasma desorption mass spectrometry (PDMS) in both negative- and positive-ion modes were determined. Negative-ion spectra gave signals for the main diphosphorylated (intact) molecular species in their native proportions. Intact and alkaline-treated lipid A in this mode gave, for the glucosamine I moiety, easily identified signals that have not been previously reported in PDMS. These spectra gave enough information to localize the fatty acids. The procedure was verified with relatively homogeneous lipids A prepared from Salmonella minnesota R595 and Neisseria meningitidis lipopolysaccharides, and then applied to the previously unstudied Yersinia entercolitica O:11,24 lipid A to obtain the localization of its fatty acids. The possibility of obtaining this much information from two negative-ion spectra was attributed to the method, described earlier, of preparing the samples. In the positive-ion mode, about half of the E. coli ions containing diglucosamine appeared as monodephosphorylated species and/or as Na adducts. The intact glucosamine II moiety and its fragment ions gave signals none of which were Na adducts. With lipids A prepared from S. minnesota, N. meningitidis, and Y. enterocolitica, similar fragmentation patterns were observed. For lipid A structure determination, the positive-ion mode could play a confirmatory role. The above results and some of those reported by others were compared.     

重组鼠疫汉坦钩端多抗原位点同源合成基因的表达及活性鉴定

将人工合成的含鼠疫耶尔森氏菌的2个抗原位点、汉坦病毒的5个抗原位点和钩端螺旋体的5个抗原位点的串联基因片段克隆到原核表达载体pET-20b中,构建重组表达质粒pET-rYHL.转化E.coli BL21(DE3)后获得表达菌株.表达菌株经IPTG诱导后,可高效表达带组氨酸标签的以包涵体形式存在的融合蛋白.包涵体蛋白经尿...     

Antioxidant, electrochemical, thermal, antimicrobial and alkane oxidation properties of tridentate Schiff base ligands and their metal complexes

In this study, two Schiff base ligands (HL(1) and HL(2)) and their Cu(II), Co(II), Ni(II), Pd(II) and Ru(III) metal complexes were synthesized and characterized by the analytical and spectroscopic methods. Alkane oxidation activities of the metal complexes were studied on cyclohexane as substrate. The ligands and their metal complexes were evaluated for their antimicrobial activity against Corynebacterium xerosis, Bacillus brevis, Bacillus megaterium, Bacillus cereus, Mycobacterium smegmatis, Staphylococcus aureus, Micrococcus luteus and Enterococcus faecalis (as gram-positive bacteria) and Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Yersinia enterocolitica, Klebsiella fragilis, Saccharomyces cerevisiae, and Candida albicans (as gram-negative bacteria). The antioxidant properties of the Schiff base ligands were evaluated in a series of in vitro tests: 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging and reducing power activity of superoxide anion radical generated non-enzymatic systems. Electrochemical and thermal properties of the compounds were investigated.     

The family of Serratia type pore forming toxins

The Serratia marcescens hemolysin represents the prototype of a growing family of pore forming toxins. The available bacterial genome sequences reveal Serratia hemolysin homologues in additional species. However, only S. marcescens hemolysin has been studied in great molecular detail. This family of toxins has nothing in common with the pore forming toxins of E. coli type (RTX toxins), the Staphylococcus aureus alpha-toxin or the thiol activated toxin of group A beta-hemolytic streptococci (Streptolysin O). Studies on erythrocytes, eukaryotic cells and artificial black lipid membranes, have shown that the mechanism of pore formation of ShlA is different form other pore forming toxins. The S. marcescens hemolysin proteins ShlB and ShlA, exhibit protein sequence homologues in Proteus mirabilis, Haemophilus ducreyi, Yersinia pestis, Yersinia enterocolitica, Edwardsiella tarda, Photorhabdus luminescens and Xylella fastidiosa . The family of Serratia type pore forming toxins show a unique secretory mechanism which has been described as a two partner secretion system (TPSS) or type V-secretion system. Not only Serratia type pore forming toxins are secreted via TPSS but also adhesins from Bordetella pertussis, Erwinia chrysanthemi and Haemophilus influenzae. The uniqueness of the Serratia family is underlined by the fact that activation of ShlA by ShlB strictly requires phosphatidylethanolamine as a cofactor. And, quite unusual, ShlA undergoes a conformational change during activation.     

毛细管电泳法快速分离和检测肠毒性大肠杆菌

 建立了快速分离和检测引起仔猪腹泻的肠毒性大肠杆菌K88、K99和 987P细菌细胞的毛细管区带电泳方法 ,并进行了腹泻仔猪粪便中肠毒性大肠杆菌的应用检测分析。结果表明 ,在电泳缓冲液为 0 0 5mol/LNa2 CO3 NaHCO3(pH 9 9)、分离电压为 1 4 1kV、检测波长为 2 1 0nm的电泳条件下 ,E coliK88、K99和 987P的细胞分别具有单一、稳定的特征谱峰 ,其保留时间的相对标准偏差RSD≤ 0 9% ;在确定的实验条件下 ,实现了腹泻仔猪粪便中肠毒性大肠杆菌的快速检测分析 ,发现将出生 5d～ 6d的腹泻仔猪粪便引入培养基中增殖后主要检出K88。     

Capture efficiency of Escherichia coli in fimbriae-mediated immunoimmobilization

Capturing pathogens on a sensor surface is one of the most important steps in the design of a biosensor. The efficiency of a biosensor at capturing pathogens has direct bearing on its sensitivity. In this work we investigated the capturing of Escherichia coli on substrates modified with antibodies targeting different types of fimbriae: K88ab (F4), K88ac (F4), K99 (F5), 987P (F6), F41, and CFA/I. The results suggest that all these fimbriae can be used for the efficient immobilization of living E. coli cells. The immobilization efficiency was affected by the purity and clone type of the antibody and the fimbriae expression level of the bacteria. For a specific fimbriae type, a higher immobilization efficiency was often observed with the monoclonal antibodies. Immunoimmobilization was utilized in an antibody microarray immersed in a mixed culture of pathogens to demonstrate the rapid and simultaneous label-free detection of multiple pathogens within less than 1 h using a single test. The capture rate of living pathogens exceeds a single bacterium per 100 × 100 μm(2) area per 0.5 h of incubation for a bulk concentration of 10(5) cfu/mL.     

两种吸附树脂对4B酸吸附行为的比较研究

研究了新型复合功能吸附树脂NDA-99和超高交联大孔吸附树脂JX-101对水溶液中4B酸(对甲苯胺-2-磺酸)的静态吸附行为和热力学特性，结果表明，NDA-99树脂对4B酸的吸附量明显大于JX-101树脂，两种树脂对4B酸的吸附均符合Freundlich吸附等温方程．其中，JX-101对4B酸的吸附属于物理吸附：NDA-99由于树脂表面存在弱碱性官能团，对4B酸的吸附表现为物理吸附和化学络合协同作用。     

Overview of the inactivation by 254 nm ultraviolet radiation of bacteria with particular relevance to biodefense

Abstract Our goal was to ultimately predict the sensitivity of untested bacteria (including those of biodefense interest) to ultraviolet (UV) radiation. In this study, we present an overview and analysis of the relevant 254 nm data previously reported and available in the literature. The amount of variability in this data prevented us from determining an "average" response for any bacterium. Therefore, we developed particular selection criteria to include the data in our analysis and suggested future guidelines for reporting UV sensitivity results. We then compiled a table of the sensitivity to 254 nm UV for 38 bacteria and three bacterial spores. The UV sensitivity was quite similar (within 10%) among the spores of Bacillus anthracis (strains Vollum 1B and Sterne), Bacillus subtilis, and Bacillus megaterium. These data indicate that spores of B. subtilis and B. megaterium could be adequate simulants of B. anthracis spores in UVC experiments. Spores of B. anthracis, B. subtilis and B. megaterium were 5-10 times more resistant to UV than were their corresponding vegetative cells. The vegetative cells of B. anthracis showed similar UV sensitivity to those of Burkholderia pseudomallei, Shigella sonnei, and a wild-type strain of Escherichia coli. Yersinia enterocolitica and Vibrio cholerae appeared more sensitive to UV and Salmonella typhi slightly more resistant to UV than E. coli. The sensitivity (at 254 nm) of all vegetative bacteria ranged from 11 to 80 Jm(2) for a 1 Log(10) kill and from 25-200 Jm(2) for 4 Log(10) kill.     

LIMITED TRYPTIC DIGESTION OF LEUCYL-tRNA SYNTHETASE AND CHARACTERIZATION OF ITS ACTIVE FRAGMENT

Leucyl-tRNA synthetase (LeuRS, EC 6.1.1.4) from E. coli underwent limitedproteolysis by trypsin which cut off 6K peptide and converted the intact LeuRS into a 96K fragment. The truncated enzyme retained the PPi exchange activity with the same kinetic parameters as those of native LeuRS but lost the tRNA~(Leu)charging, binding and other tRNA~(Leu)-related activities. N-terminus analysis showed that the 6K peptide was located at the C-terminus of LeuRS. This small part played a crucial role in tRNA~(Leu) binding. Our results suggest that the two activities, PPi exchange and tRNA charging are independent of each other and correspond to different structural regions of LeuRS. The C-terminal region might be the tRNA~(Leu)binding site of LeuRS.     

CLONING, EXPRESSION AND CHARACTERIZATION OF Pro3 GENE OF YEAST Saccharomyces cerevisiae

The proline biosynthetic pathway and Pro genes in Saccharomyces cerevisiae have just begun to be studied recently. In our laboratory, Pro2 gene of S. cerevisiae had been cloned in yeast. As described in this paper, yeast Pro3 gene was also cloned, which can complement yeast Pro3 mutants, and be expressed efficiently in E. coli. The high activities of this gene product, L-pyrroline-5-carboxylate (P5C) reductase, can be detected in both organisms. The activity of the Pro3 gene product in multiple copy plasmids is not higher than that of single copy genes in chromosomes in both yeast and E. coll. The preliminary characterization of the gene is also reported.     

THE EXPRESSION OF ENTEROTOXIN A~-B~+ GENE OF V. cholerae IN E. coli

The cloning in E. coli of a cholerae toxin gene that is A~-B~+ has been successfully constructed by using DNA recombinant techniques. E. coli cells carrying the recombinant plasmid pMM-CTB have been shown to produce a large amount of CTB subunits which are secreted as extracellular proteins.     

Effect of extraction conditions on measured total polyphenol contents and antioxidant and antibacterial activities of black tea

Black tea was extracted for 2, 8 and 18 h with absolute acetone, N,N-dimethyl-formamide (DMF), ethanol and methanol and their 50% aqueous solutions. The extracts were screened for total polyphenol contents, antioxidant and antibacterial activities. The polyphenol content of the extracts was found to be in the range of 0.44-114.01 mg gallic acid equivalents (GAE)/g dry weight tea, depending on the solvent used and the length of the extraction process. In general, aqueous acetone or DMF extracts displayed the highest polyphenol contents and antioxidant activity, while absolute acetone was the least efficient solvent. Antioxidant activities of tea extracts tested using the reducing power and 2,2-diphenyl-1-picryhydrazyl (DPPH) radical methods ranged from 0.09 to 1.18 and from 2.60 to 95.42 %, respectively, depending on the extraction conditions and the antioxidant activities correlated well with the polyphenol concentrations. Aqueous solvent black tea extracts also possessed antibacterial activity, depending on the solvent used and bacterial species tested. Staphylococcus aureus was found to be the most sensitive to all tea extracts, except for the methanol extract. Tea extracts were not effective against Y. enterocolitica, L. monocytogenes and E. coli O157:H7.     

Profiling the structural determinants for the selectivity of representative factor-Xa and thrombin inhibitors using combined ligand-based and structure-based approaches

The current study deciphers the combined ligand- and structure-based computational insights to profile structural determinants for the selectivity of representative diverse classes of FXa-selective and thrombin-selective as well as dual FXa-thrombin high affinity inhibitors. The thrombin-exclusive insertion 60-loop (D-pocket) was observed to be one of the most notable recognition sites for the known thrombin-selective inhibitors. Based on the topological comparison of four common active-site pockets (S1-S4) of FXa and thrombin, the greater structural disparity was observed in the S4-pocket, which was more symmetrical (U-shaped) in FXa as compared to thrombin mainly due to the presence of L99 and I174 residues in latter in place of Y99 and F174 respectively in former protease. The S2 pocket forming partial roof at the entry of 12 ? deep S1-pocket, with two extended β-sheets running antiparallel to each other by undergoing U-turn (～180?), has two conserved glycine residues forming H-bonds with the bound ligand for governing ligand binding affinity. The docking, scoring, and binding pose comparison of the representative high-affinity and selective inhibitors into the active sites of FXa and thrombin revealed critical residues (S214, Y99, W60D) mediating selectivity through direct- and long-range electrostatic interactions. Interestingly, most of the thrombin-selective inhibitors attained S-shaped conformation in thrombin, while FXa-selective inhibitors attained L-shaped conformations in FXa. The role of residue at 99th position of FXa and thrombin toward governing protease selectivity was further substantiated using molecular dynamics simulations on the wild-type and mutated Y99L FXa bound to thrombin-selective inhibitor 2. Furthermore, predictive CoMFA (FXa q2 = 0.814; thrombin q2 = 0.667) and CoMSIA (FXa q2 = 0.807; thrombin q2 = 0.624) models were developed and validated (FXa r2(test) = 0.823; thrombin r(2)(test) = 0.816) to feature molecular determinants of ligand binding affinity using the docking-based conformational alignments (DBCA) of 141 (88(train)+53(test)) and 39 (27(train)+11(test)) nonamidine class of potent FXa (0.004 ≤ K(i) (nM) ≤ 4700) and thrombin (0.001 ≤ K(i) (nM) ≤ 940) inhibitors, respectively. Interestingly, the ligand-based insights well corroborated with the structure-based insights in terms of the role of steric, electrostatic, and hydrophobic parameters for governing the selectivity for the two proteases. The new computational insights presented in this study are expected to be valuable for understanding and designing potent and selective antithrombotic agents.     

微量热法测定细菌的临界生长温度

应用微量热学的方法,我们已能成功地测得细菌生长过程的热谱,这种热谱包含着有关细菌生长代谢过程的丰富信息,例如对热谱曲线的指数生长段进行解析,可得出细菌生长的速率常数、激活能和有关的热力学参数。故采用微量热法测定细菌在不同培养温度下的生长速率常数,利用计算机拟合出相应k(速率常数)和T(培养温度)的线性关系式后,若把生长速率为零的温度定义为临界生长温度时,就可以根据由上述实验所得的k～T线性关系式求得细菌的临界生长温度。本工作仍采用微量热法对福氏志贺氏菌(S. flexneri)和大肠埃希氏菌(E. coli)进行实验测定。按文献的方法求出它们在不同温度下的生长速率常数;对于大肠埃希氏菌还用几种不同的培养基分别进行实验测定。     

稀土(Y、Ce、Sm)对Ni-P非晶态合金热稳定性的影响

用差示扫描量热法(DSC)研究了非晶态合金Ni-RE-P(RE=Y, Ce, Sm,下同)的热稳定性; 用X-光衍射(XRD)和扫描电镜(SEM)检测了在不同温度范围处理的样品的结构变化. 结果表明, 向非晶态Ni-P合金中加入少量稀土元素(Y, Ce, Sm), 可显著提高非晶态Ni-RE-P合金的热稳定性.样品的晶化激活能数据表明, Ni-RE-P的各转变阶段的激活能都比Ni-P的大, 说明Ni-RE-P比Ni-P更难晶化, 即Ni-RE-P比Ni-P更稳定.