Production of 8'-halogenated and 8'-unsubstituted novobiocin derivatives in genetically engineered streptomyces coelicolor strains

In the present study, we produced a hybrid antibiotic, carrying a chlorine atom instead of a methyl group at position 8 of the aminocoumarin moiety of novobiocin. This compound was not accessible by conventional gene inactivation/gene expression experiments due to difficulties in the genetic manipulation of the novobiocin producer Streptomyces spheroides. However, the desired compound was obtained after modification of the novobiocin biosynthetic gene cluster by using lambda-Red-mediated recombination in Escherichia coli, followed by integration of the resulting modified cosmid into the phiC31 attachment site of Streptomyces coelicolor and coexpression of the halogenase Clo-hal of clorobiocin biosynthesis. The halogenase BhaA, responsible for chlorination of tyrosyl moieties of the glycopeptide antibiotic balhimycin, was unable to functionally replace the halogenase Clo-hal, suggesting that the two enzymes have different substrate specificities.     

Immobilization of E. coli with autodisplayed Z-domains to a surface-modified microplate for immunoassay

Escherichia coli with autodisplayed Z-domains was reported to improve the sensitivity of immunoassays by the orientation control of antibodies. In this work, a sensitive microplate-based immunoassay is presented by immobilizing E. coli cells to a surface-modified microplate. The microplate was prepared by coating parylene-H film with formyl groups, and then covalently coupling poly-L-lysine to the parylene-H film. The E. coli cells were bound to the microplate by charge interactions between the negatively charged E. coli outer membrane and the positively charged microplate surface. In this work, the preparation of the microplate coated with poly-L-lysine is presented. The immobilization efficiency of E. coli to the modified surface was estimated to be far higher than non-specific interaction by fluorescence microscope and the optical transmittance of the modified microplate was measured to be feasible for immunoassay. The microplate-based immunoassay is demonstrated to be feasible for medical diagnosis of inflammatory diseases by using C-reactive protein as a target analyte for the medical diagnosis of inflammatory diseases.     

Construction and evaluation of nagR-nagAa::lux fusion strains in biosensing for salicylic acid derivatives

The NagR protein is a response regulatory protein found in the bacterium Ralstonia sp. U2 that is involved in sensing for salicylic acid and the subsequent induction of the operon just upstream of its gene. The genes encoded for in this operon are involved in the degradation of salicylic acid. Escherichia coli strain RFM443 carrying a fusion of the Photorhabdus luminescens luxCDABE operon with the nagR gene and upstream region of the nagAa gene was constructed and characterized with respect to its optimum temperature, its response time and kinetics, and its ability to detect numerous benzoic acid derivatives. Although capable of detecting 0.5 mM salicylic acid at any temperature between 28 and 40 degrees C, this E. coli strain, labeled DNT5, showed its greatest relative activity at 30 degrees C, i.e., the temperature at which the largest induction was seen. Furthermore, experiments done with numerous benzoic acid derivatives found the NagR protein to be responsive to only a few of the compounds tested, including salicylic acid and 3-methyl salicylic acid, and acetyl salicylic acid was the strongest inducer. The lower limits of detection for these compounds with E. coli strain DNT5 were also established, with the native inducer, salicylic acid, giving the most sensitive response and detectable down to a concentration of about 2 microM. A second lux fusion plasmid was also constructed and transformed into an NahR background, Pseudomonas putida KCTC1768. Within this strain, NAGK-1768, the supplemental activity of the NahR protein on the nagAa promoter, was shown to extend both the range of chemicals detected and the sensitivity.     

Crystallographic and spectroscopic evidence for high affinity binding of FeEDTA(H2O)- to the periplasmic nickel transporter NikA

Because nickel is both essential and toxic to a great variety of organisms, its detection and transport is highly regulated. In Escherichia coli and other related Gram-negative bacteria, high affinity nickel transport depends on proteins expressed by the nik operon. A central actor of this process is the periplasmic NikA transport protein. A previous structural report has proposed that nickel binds to NikA as a pentahydrate species. However, both stereochemical considerations and X-ray absorption spectroscopic results are incompatible with that interpretation. Here, we report the 1.8 A resolution structure of NikA and show that it binds FeEDTA(H2O)- with very high affinity. In addition, we provide crystallographic evidence that a metal-EDTA complex was also bound to the previously reported NikA structure. Our observations strongly suggest that nickel transport in E. coli requires the binding of this metal ion to a metallophore that bears significant resemblance to EDTA. They also provide a basis for the potential use of NikA in the bioremediation of toxic transition metals and the design of artificial metalloenzymes.     

Rapid and ultrasensitive E. coli O157:H7 quantitation by combination of ligandmagnetic nanoparticles enrichment with fluorescent nanoparticles based two-color flow cytometry

A novel, fast and sensitive determination strategy for E. coli O157:H7 has been developed by combination of ligandmagnetic nanoparticles (LMNPs) enrichment with a fluorescent silica nanoparticles (FSiNPs) based two-color flow cytometry assay (LMNPs@FSiNPs-FCM). E. coli O157:H7 was first captured and enriched through the lectin concanavalin A (Con A) favored strong adhesion of E. coli O157:H7 to the mannose-conjugated magnetic nanoparticles. The enriched E. coli O157:H7 was further specially labeled with goat anti-E. coli O157:H7 antibody modified RuBpy-doped FSiNPs, and then stained with a nucleic acid dye SYBR Green I (SYBR-I). After dual-labeling with FSiNPs and SYBR-I, the enriched E. coli O157:H7 was determined using multiparameter FCM analysis. With this method, the detection sensitivity was greatly improved due to the LMNPs enrichment and the signal amplification of the FSiNPs labelling method. Furthermore, the false positives caused by aggregates of FSiNPs conjugates and nonspecific binding of FSiNPs to background debris could be significantly decreased. This assay allowed the detection of E. coli O157:H7 in PB buffer at levels as low as 7 cells mL(-1). The total assay time including E. coli O157:H7 sample enrichment and detection was less than 4 h. An artificially contaminated bottled mineral water sample with a concentration of 6 cells mL(-1) can be detected by this method. It is believed that the proposed method will find wide applications in biomedical fields demanding higher sensitive bacterial identification.     

纳米Ag2O2-PbO2化学修饰电极对大肠杆菌细胞膜壁和DNA损伤的研究

制备了一种新型纳米AgO2-PbO2修饰电极,在选定的正电位下,电极表面产生羟基自由基(.OH).通过测定产生的脂质过氧化物和漏出蛋白质的量来研究羟基自由基对大肠杆菌细胞膜壁损伤的情况,并运用电泳和DNA测序的方法对大肠杆菌质粒DNA的损伤及其对序列变化进行了研究.     

Au纳米标记物增强电化学免疫分析大肠杆菌的研究

通过在Au纳米颗粒表面修饰辣根过氧化酶(HRP)标记的大肠杆菌抗体制备了一种新型的Au纳米标记物, 并将该纳米标记物应用于增强电化学免疫分析大肠杆菌. 经过酶联免疫反应后, Au纳米标记物、免疫磁性颗粒(IMB)和大肠杆菌形成了IMB/抗体-大肠杆菌-Au纳米标记物的三明治式免疫复合物. 以3,3,5,5-四甲基联苯二胺(TMB)溶液作为底物, 采用电化学与流动注射检测(FIA)相结合的技术测定HRP的活性. 检测到的电流大小与免疫复合物上HRP的量成正比, 从而与大肠杆菌的浓度成正比. Au纳米颗粒增加了HRP的负载量, 增强了电化学信号, 大大提高了大肠杆菌的检测灵敏度. 实验结果表明, 大肠杆菌浓度在 1.0×102～5.0×104 cfu•mL－1范围内与电流大小成线性相关, 最低检测限达50 cfu•mL－1, 若对大肠杆菌样品溶液进行预浓缩, 将得到更宽的检测范围和更低的检测限. 本方法总的分析时间比其他方法短, 在1 h内就能完成对大肠杆菌样品的快速检测.     

Clorobiocin biosynthesis in Streptomyces: identification of the halogenase and generation of structural analogs

Clorobiocin (clo) and novobiocin (nov) are potent inhibitors of bacterial DNA gyrase. The two substances differ in the substitution pattern at C-8' of the aminocoumarin ring, carrying a chlorine atom or a methyl group, respectively. By gene inactivation, clo-hal was identified as the gene of the halogenase responsible for the introduction of the chlorine atom of clorobiocin. Inactivation of cloZ did not affect clorobiocin formation, showing that this ORF is not essential for clorobiocin biosynthesis. Expression of the methyltransferase gene novO in the clo-hal(-) mutant led to the very efficient formation of a hybrid antibiotic containing a methyl group instead of a chlorine atom at C-8'. Comparison of the antibacterial activity of clorobiocin analogs with -Cl, -H, or -CH(3) at C-8' showed that chlorine leads to 8-fold higher activity than hydrogen and to 2-fold higher activity than a methyl group.     

Fragmentation of a novel marine peptide, plicatamide, involves an unusual gas-phase intramolecular rearrangement

During our characterization of plicatamide 1, a modified octapeptide: Phe-Phe-His-Leu-His-Phe-His-dc deltaDOPA (where dc deltaDOPA = decarboxy-(E)-alpha,beta-dehydro-3,4-dihydroxyphenylalanine) from the blood cells of the ascidian Styela plicata, we noted a series of fragment ions from the [M + H]+ ion which could not be assigned. There was no evidence in the 1H NMR spectrum to support an alternative molecular structure and the series of fragment ions were not present in the tandem mass spectrometry analysis of the [M + Na]+ ion. In addition, there was no evidence that the sample was a mixture of isobaric compounds. We propose that an unusual C-terminal to N-terminal rearrangement is responsible for the series of fragment ions from the [M + H]+ ion. This rearrangement was not observed in peptide analogs of plicatamide which did not contain the dc deltaDOPA at the C-terminus suggesting that this moiety is critical for the rearrangement. The proposed reaction is analogous to that recently reported by Vachet et al. involving a fragment ion formed from leucine enkephalin.     

Escherichia coli O86 O-antigen biosynthetic gene cluster and stepwise enzymatic synthesis of human blood group B antigen tetrasaccharide

Previous study showed that some Gram-negative bacteria possess human blood group activity. Among them, Escherichia coli O86 has high blood group B activity and weak blood group A activity. This is due to the cell surface O-antigen structure, which resembles that of human blood group B antigen. In this study, we sequenced the entire E. coli O86 antigen gene cluster and identified all the genes responsible for O-antigen biosynthesis by sequence comparative analysis. The blood group B-like antigen in E. coli O86 O-polysaccharide was synthesized by sequentially employing three glycosyltransferases identified in the gene cluster. More importantly, we identified a new bacterial glycosyltransferase (WbnI) equivalent to human blood group transferase B (GTB). The enzyme substrate specificity and stepwise enzymatic synthesis of blood group B-like antigen revealed that the biosynthetic pathway of B antigen is essentially the same in E. coli O86 as in humans. This new finding provides a model to study the specificity and structure relationship of blood group transferases and supports the hypothesis of anti-blood group antibody production by bacterial stimulation.     

Sensitivity and specificity of the test kit BAX for screening/E. coli O157:H7 in ground beef: independent laboratory study

An independent laboratory study of the BAX for Screening/E. coli O157:H7 kit was conducted at the National Food Laboratory, Inc., Dublin, CA, to complete AOAC Performance Tested Method certification. The BAX system kit was compared with the BAM culture method and a modified BAM culture method for detection of E. coli O157:H7 in ground beef. The BAX system kit detected the target organism at levels approximately 10-fold lower than those that gave positive BAM results. This study validated product claims, and Performance Tested Method status was granted.     

Identification of the binding of sceptrin to MreB via a bidirectional affinity protocol

A bidirectional affinity system was used to screen for marine natural products that bound to Escherichia coli proteins. A system was developed and applied to isolate the natural product sceptrin from an Agelas conifera extract and its affinity partner MreB from E. coli lysate. The use of a dual immunoaffinity fluorescent (IAF) tag permitted this process to co-immunoprecipitate the bacterial equivalent of actin, MreB, from E. coli lysate. MreB was subsequently validated as a target for sceptrin using a resistance mapping approach. The combination of these studies suggests that natural products and their protein targets can be isolated in concert using a melody of forward and reverse affinity matrices. While the structure of sceptrin was elucidated by NMR analysis, the bulk of effort was conducted without knowing the structure of the natural product, thereby elevating a key bottleneck in the development of high-throughput methods for natural product discovery.     

Effect of Escherichia coliform on the biomineralization of calcium bilirubinate in mimic systems

The influence of Escherichia coliform (E. coli), especially the proteins it secretes, on the nucleation, growth and aggregation processes of calcium bilirubinate (CaBR) in different mimic systems, such as NaCl aqueous solution and model bile, is studied in this paper. The results show that in NaCl aqueous solution the morphology of calcium bilirubinate (CaBR) changes from amorphous sheet structure to a highly self-organized and highly self-replicated fractal structure that is accumulated by rhombic microcrystals after the addition of E. coli. In model bile with the existence of E. coli, CaBR also forms a fractal structure, but the fractal structure is staghorn-like. Meanwhile, the composition of the prepared CaBRs is nonstoichiometric, and the crystallization is greatly improved with the existence of E. coli. Besides, formation mechanism of CaBRs affected by E. coli is explored, showing that the proteins secreted by E. coli interact with the Ca(2+) ions to provide nucleation sites for CaBRs, and the conformation of the proteins becomes more ordered, resulting in the improvement of the crystalline of CaBRs. On the other hand, the interaction of proteins and the Ca(2+) ions also promote the aggregation state of the CaBRs.     

通过氧化改性提高载银活性炭的抗菌性能

使用浓HNO3和浓H2O2对活性炭进行常温氧化改性,用FTIR和N2吸附法对活性炭进行表面分析,用AAS、SEM、XRD研究银在活性炭表面的吸附和分布特征,并研究了载银活性炭的抗茵性能.结果表明,活性炭经浓HNO3常温改性后,比表面积提高,而经浓H2O2常温改性后,比表面积略有下降,但都使活性炭表面含氧基团增加.改性后,活性炭表面增加的含氧基团为[Ag(NH3)2] 的还原吸附提供更多的活性点,使银的吸附量增大5倍多,银颗粒更加密集,大小更加均一.研究表明,载银活性炭具有明显的抗茵作用,其中对金黄色葡萄球菌的杀灭效果优于对大肠杆菌的杀灭效果,氧化改性使载银活性炭抗茵作用显著增强,其中硝酸改性现象更加明显.     

基于Fe3O4@Au复合纳米粒子标记抗体的电化学免疫方法用于水体中大肠杆菌的检测

合成了Fe₃O₄@Au复合纳米粒子作为辣根过氧化酶标记抗体的载体, 并将该复合纳米粒子标记物应用于电化学放大免疫分析. 将电子媒介体硫堇聚合在玻碳电极表面, 以纳米金作为固定大肠杆菌抗体的基底, 通过辣根过氧化酶催化溶液中H₂O₂产生的电流信号来测定大肠杆菌. 实验结果表明, 该方法对水体中大肠杆菌检测的线性范围为50～1×10⁵ cfu/mL, 检出限为20 cfu/mL. 对过富集后的实际水样进行测定, 该法结果表明, 对水体中大肠杆菌的检测灵敏度达到2 cfu/mL.     

通过柠檬酸改性提高载银活性炭的抗菌性能

通过负载柠檬酸对活性炭进行改性,用N2吸附法测定活性炭的比表面积,用AAS、SEM、XRD测试技术分析了银在活性炭上的吸附和分布,并研究了载银活性炭的抗菌性能。结果表明,负载柠檬酸使活性炭的比表面积下降约24%,但载银后活性炭的比表面积增大。柠檬酸改性为[Ag(NH3)2] 的还原吸附提供更多的活性点,使银的吸附速率加快,吸附量提高约25%,表面的银颗粒变得非常密集,粒径减小,且颗粒均匀,因此抗菌性能显著增强,其中对金黄色葡萄球菌的杀灭效果明显优于对大肠杆菌的,同时对于高分散Ag/C催化剂的制备及银的回收也具有重要的价值。     

碱性品红修饰磁性纳米粒子的制备及在分枝杆菌分离中的应用

痰或其它各种体液内结核分枝杆菌(MTB)阳性是诊断结核病(TB)的金标准.结核硬脂酸(TBSA)是鉴定分枝杆菌与其它非分枝杆菌的特征组分,可用于TB的诊断[1,2]。     

Comparative validation study to demonstrate the equivalence of a minor modification to AOAC Method 996.09 [Visual Immunoprecipitate (VIP) for E. coli O157:H7] to the reference culture methods

The Visual Immunoprecipitate (VIP) for the Detection of E. coli O157:H7 in Foods, AOAC Official Method 996.09, has been modified to use a simplified plastic housing for the device. A methods comparison study was conducted to demonstrate the equivalence of this modification to the reference culture method. Three foods were analyzed. In total, valid results were obtained from 240 samples and controls. Results showed that the VIP for E. coli O157:H7 is equivalent to the reference culture methods for the detection of E. coli O157:H7.