Ultrahigh-throughput screening to identify E. coli cells expressing functionally active enzymes on their surface

We show here that E. coli bacteria that display esterases or lipases on their cell surface together with horseradish peroxidase (HRP) are capable of hydrolysing carboxylic acid esters of biotin tyramide. The tyramide radicals generated by the coupled lipase-peroxidase reaction were short-lived and therefore became covalently attached to reactive tyrosine residues that were located in close vicinity on the surface of a bacterial cell that displayed lipase activity. Up to 120 000 biotinylated tyramide derivatives could be covalently coupled through HRP activation to the surface of a single living E. coli cell. Differences in cellular esterase activity were found to correlate with the amount of biotin tyramide deposited on the cell surface. Selective biotin tyramide labelling of cells that had lipase activity allowed their isolation by magnetic cell sorting from a 1:10(6) mixture of control cells. This strategy of covalently attaching a biotin label to esterase-proficient bacteria might open new avenues to ultrahigh-throughput screening of enzyme libraries for hydrolytic enzymes with enhanced activities or enantioselectivities.     

On the role of conserved proline residues in the structure and function of Clostridium pasteurianum 2[4Fe-4S] ferredoxin

The widespread occurrence of Pro residues adjacent to Cys ligandsin the sequences of [4Fe-4S] electron transfer proteins hasnot yet found a functional basis. The two such Pro of Clostridiumpasteurianum 2[4Fe-4S] ferredoxin have been probed by site-directedmutagenesis. Any one of them, but not both simultaneously, canbe substituted without impairing the proper folding of the protein.The reduction potentials of the ferredoxin variants fall ina narrow range of <20 mV above the potential of the nativeprotein. The biological activities with C.pasteurianum hydrogenaseand pyruvate-ferredoxin oxidoreductase do not change significantly,except when Lys replaces Pro. In these cases, the data suggestthat the two clusters of 2[4Fe-4S] ferredoxin may not alwaysbe equivalent in the interaction with the redox partners. Destabilizationof the structure has been observed as the consequence of theProl9 or Pro48 substitutions. Using 2-D NMR, this effect hasbeen associated with perturbations of both the hydrogen bondnetwork and one amino acid side chain around the [4Fe-4S] clusters.Thus, the conserved Pro found in the binding motif of [4Fe-4S]clusters in proteins strongly stabilizes the active site butdoes not play an essential role in the mechanism of electrontransfer.     

The [4Fe-4S]-Cluster of HydF is not Required for the Binding and Transfer of the Diiron Site of [FeFe]-Hydrogenases

The active site of [FeFe]-hydrogenases contains a cubane [4Fe-4S]-cluster and a unique diiron cluster with biologically unusual CO and CN⁻ ligands. The biogenesis of this diiron site, termed [2Fe_H], requires the maturation proteins HydE, HydF and HydG. During the maturation process HydF serves as a scaffold protein for the final assembly steps and the subsequent transfer of the [2Fe_H] precursor, termed [2Fe_P], to the [FeFe]-hydrogenase. The binding site of [2Fe_P] in HydF has not been elucidated, however, the [4Fe-4S]-cluster of HydF was considered as a possible binding partner of [2Fe_P]. By targeting individual amino acids in HydF from Thermosipho melanesiensis using site directed mutagenesis, we examined the postulated binding mechanism as well as the importance and putative involvement of the [4Fe-4S]-cluster for binding and transferring [2Fe_P]. Surprisingly, our results suggest that binding or transfer of [2Fe_P] does not involve the proposed binding mechanism or the presence of a [4Fe-4S]-cluster at all.     

利用离子液体[BMIM]2[CR]萃取牛血清蛋白的研究

利用离子交换过程将亲和性染料刚果红合成为[BMIM]2[CR],并进行牛血清蛋白萃取实验。研究结果表明,[BMIM]2[CR]离子液体的浓度为10mmol／L,萃取时间为30min以及溶液呈酸性时,对牛血清蛋白的萃取效果最佳,可达90％以上。该研究可为[BMIM]2[CR]实际蛋白质萃取应用中提供一定的可行性条件。     

应用RED同源重组技术构建表面展示链球菌GapC1的大肠埃希菌

目的应用RED同源重组技术构建表面展示链球菌Gap C1-150 aa(Gap C1)的大肠埃希菌,为进一步构建大肠埃希菌表面展示重组菌株奠定基础。方法根据Gen Bank中登录的gap C基因序列(GI:30348860)设计引物,以质粒p KD3、p QE30/lpp-omp A-gap C为模板进行PCR扩增、融合,获得融合片段,转化入含p KD46质粒的大肠埃希菌HB101中,利用p KD46编码的RED系统将融合片段重组入基因组中,经氨苄西林(Amp)和氯霉素(Cm)抗性筛选,获得去除p KD46质粒的重组菌(HB101LOG)。对重组菌株进行PCR、Western blot、流式细胞术、荧光显微镜及生物学特性检测。结果 PCR结果显示,重组菌株构建正确;重组大肠埃希菌HB101LOG在菌体表面表达了外源蛋白Gap C1-150 aa;激光共聚焦显微镜观察到HB101LOG可见明显的绿色荧光,HB101未见绿色荧光;重组菌株HB101LOG和HB101的生长曲线符合大肠埃希菌的生长规律。结论应用RED系统成功构建了能展示链球菌Gap C1-150 aa的大肠埃希菌重组菌株。     

Reply to ``Transesterification on imprinted silica' [by W.R. Ahmad and M.E. Davis]

No abstract available This revised version was published online in July 2006 with corrections to the Cover Date.     

Inactivation of E. coli,S. aureus,and Bacteriophages in Biofilms by Humidified Air Plasma

In this study, humidified air dielectric barrier discharge (DBD) plasma was used to inactivate Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), and bacteriophages in biofilms containing DNA, NaCl, carbohydrates, and proteins. The humidified DBD plasma was very effective in the inactivation of microbes in the (≤1.0 μm) biofilms. The number of surviving E. coli, S. aureus, and bacteriophages in the biofilms was strongly dependent on the constituent and thickness of the biofilms and was greatly reduced when the plasma treatment time increased from 5 s to 150 s. Our analysis shows that the UV irradiation was not responsible for the inactivation of microbes in biofilms. The short-lived RONS generated in the humidified air DBD plasma were not directly involved in the inactivation process; however, they recombined or reacted with other species to generate the long-lived RONS. Long-lived RONS diffused into the biofilms to generate very active species, such as ONOOH and OH. This study indicates that the geminated NO₂ and OH pair formed due to the homolysis of ONOOH can cause the synergistic oxidation of various organic molecules in the aqueous solution. Proteins in the biofilm were highly resistant to the inactivation of microbes in biofilms, which is presumably due to the existence of the unstable functional groups in the proteins. The unsaturated fatty acids, cysteine-rich proteins, and sulfur–methyl thioether groups in the proteins were easily oxidized by the geminated NO₂ and OH pair.     

利用离子液体[BMIM]2[CR]萃取牛血清蛋白的研究

利用离子交换过程将亲和性染料刚果红合成为[BMIM]2[CR],并进行牛血清蛋白萃取实验。研究结果表明,[BMIM]2[CR]离子液体的浓度为10 mmol/L,萃取时间为30 min以及溶液呈酸性时,对牛血清蛋白的萃取效果最佳,可达90%以上。该研究可为[BMIM]2[CR]实际蛋白质萃取应用中提供一定的可行性条件。     

Effect of chlorphentermine on incorporation of [14C]choline in the rat lung phospholipids

The effect of chlorphentermine (CP) treatment (50 mg/kg/ day, per os [po]) on the incorporation of [¹⁴C]choline into rat lung phospholipid was studied.Total phospholipid content was increased 2.0-fold and 1.7-fold after seven and 14 days, respectively, compared with the pair-fed rats. The incorporation of [¹⁴C]choline into phosphatidylcholine (PC) was significantly inhibited by either seven or 14 days of CP treatment. Nevertheless, the PC content was significantly increased by day 7 and stayed elevated at day 14 of CP treatment. Choline and phosphorylcholine contents were significantly decreased by the CP treatment. These results suggest that the higher accumulation of PC is due to inhibition of enzymes involved in the hydrolysis of phospholipids rather than to a stimulation of the phospholipid synthesis. Presented in part at the SOT Meeting, Atlanta, GA, March 1984 (abstracted inThe Toxicologist 4[1], 64).     

Binding of helix-threading peptides to E. coli 16S ribosomal RNA and inhibition of the S15-16S complex

Helix-threading peptides (HTPs) constitute a new class of small molecules that bind selectively to duplex RNA structures adjacent to helix defects and project peptide functionality into the dissimilar duplex grooves. To further explore and develop the capabilities of the HTP design for binding RNA selectively, we identified helix 22 of the prokaryotic ribosomal RNA 16S as a target. This helix is a component of the binding site for the ribosomal protein S15. In addition, the S15-16S RNA interaction is important for the ordered assembly of the bacterial ribosome. Here we present the synthesis and characterization of helix-threading peptides that bind selectively to helix 22 of E. coli 16S RNA. These compounds bind helix 22 by threading intercalation placing the N termini in the minor groove and the C termini in the major groove. Binding is dependent on the presence of a highly conserved purine-rich internal loop in the RNA, whereas removal of the loop minimally affects binding of the classical intercalators ethidium bromide and methidiumpropyl-EDTAFe (MPEFe). Moreover, binding selectivity translates into selective inhibition of formation of the S15-16S complex.     

Uptake of [14C] choline and incorporation into lung phospholipid by the isolated perfused rat lung

We have used the isolated perfused lung (IPL) preparation from the rat to determine whether uptake of choline from the vascular compartment could limit the rate of synthesis of phosphatidylcholine (PC). The uptake of choline was rapid and did not saturate at a concentration of 10 mM. The rate of incorporation of choline into phospholipid was saturated above 0.1 mM choline. Whereas, uptake and incorporation were depressed at 4 C, uptake was neither dependent on the extracellular sodium concentration nor inhibited by equimolar concentrations of hemicholinium-3 (HC-3). We could find no evidence that uptake might limit synthesis of lung lecithin and conclude that uptake is either by free diffusion, or by a carrier-mediated process with a very high Km.     

Suzuki Cross-Coupling Reaction Catalyzed by the Palladium Complex Pd[N-MorphC(S)NP(O)(OiPr)2-O,S]2

Suzuki cross-coupling reaction between phenyl bromide and phenylboronic acid, catalyzed by the palladium complex Pd[N-MorphC(S)NP(O)(OiPr)₂-1,5-O,S)]₂ in acetonitrile, toluene, THF or DMF has been investigated. Bases employed for the reaction were Na₂CO₃, K₂CO₃ or Cs₂CO₃. Varying largely the experimental conditions we found that excellent yields of the product were obtained using toluene and K₂CO₃ at 100 °C at the catalyst amount of 0.02 mmol.     

Catalytic transfer hydrogenation of 2-butanone on MgO. New active surface sites generated by treatment with chloroform

In the course of heterogeneous catalytic transfer hydrogenation (CTH) of carbonyl compounds with alcohols on MgO, the catalyst is continuously poisoned, which leads to a complete loss of activity. The main result of the present work is that deactivation may be prevented by treatment with chloroform and the initial activity of MgO is retained for as long as 65 h. Detailed investigations were carried out (activations of MgO, TG, DSC, chloroform treatment, FT-IR spectroscopy, CDCl₃ adsorption) in order to identify both types of active centers responsible for poisoning and CTH. Based on our own experimental results as well as on data in the literature, the prerequisites of CTH on MgO appear to be basic (O^2–) site and adjacent surface hydroxyl site pairs. Lewis acid centers responsible for poisoning are blocked and active surface hydroxyl groups of weakly acidic character are simultaneously generated by chloroform treatment.     

二维纳米发光材料[CdxMg6-xAl2(OH)16]2+[S2-·4H2O]2-:Eu3+发光性质的研究

报道了稀土离子Eu3+的含量对二维纳米发光材料[CdxMg6-xAl2(OH)16]2+[S2-@4H2O]2-发光强度的影响,并对基质中Cd2+离子的含量、硫化时间、灼烧温度等合成条件对发光材料发光强度的影响进行了研究.     

Chemical rescue of active site mutants of S. pneumoniae surface endonuclease EndA and other nucleases of the HNH family by imidazole

The His-Asn-His (HNH) motif characterizes the active sites of a large number of different nucleases such as homing endonucleases, restriction endonucleases, structure-specific nucleases and, in particular, nonspecific nucleases. Several biochemical studies have revealed an essential catalytic function for the first amino acid of this motif in HNH nucleases. This histidine residue was identified as the general base that activates a water molecule for a nucleophilic attack on the sugar phosphate backbone of nucleic acids. Replacement of histidine by an amino acid such as glycine or alanine, which lack the catalytically active imidazole side chain, leads to decreases of several orders of magnitude in the nucleolytic activities of members of this nuclease family. We were able, however, to restore the activity of HNH nuclease variants (i.e., EndA (Streptococcus pneumoniae), SmaNuc (Serratia marcescens) and NucA (Anabaena sp.)) that had been inactivated by His→Gly or His→Ala substitution by adding excess imidazole to the inactive enzymes in vitro. Imidazole clearly replaces the missing histidine side chain and thereby restores nucleolytic activity. Significantly, this chemical rescue could also be observed in vivo (Escherichia coli). The in vivo assay might be a promising starting point for the development of a high-throughput screening system for functional EndA inhibitors because, unlike the wild-type enzyme, the H160G and H160A variants of EndA can easily be produced in E. coli. A simple viability assay would allow inhibitors of EndA to be identified because these would counteract the toxicities of the chemically rescued EndA variants. Such inhibitors could be used to block the nucleolytic activity of EndA, which as a surface-exposed enzyme in its natural host destroys the DNA scaffolds of neutrophil extracellular traps (NETs) and thereby allows S. pneumoniae to escape the innate immune response.     

Influence of surface chemical structure of active carbon on its electrochemical behaviour in the presence of silver

Cyclic voltammetric studies of the influence of surface chemistry on the electrochemical behaviour of powdered active carbon electrodes were carried out in the presence of silver both in bulk solution and pre-adsorbed on carbon. Surfaces with a variety of chemical properties were obtained through the modification of carbon samples by heat treatment under vacuum, oxidation with concentrated nitric acid and annealing in an ammonia atmosphere. The surface area (BET), acid–base neutralization capacities and sorption capacity towards Ag+ ions were estimated in the samples obtained. The states and relative amounts of deposited Ag species were estimated using XPS. The influence of the surface chemistry of the powdered carbon material in terms of its electrochemical properties and the mechanism of silver adsorption and deposition is discussed.     

Second-sphere Coordination of [Pt(bipy)(NH3)2]2+ by Dibenzo-crown Ethers. Solution Spectroscopic Studies and the Crystal and Molecular Structures of [Pt(bipy)(NH3)2.dibenzo-30-crown-10] [PF6]2.0.6 H2O and [Pt(bipy)(NH3)2.dibenzo-24-crown-8] [PF6]2

In addition to hydrogen bonding and Coulombic forces, charge transfer interactions stabilise the 1:1 adducts formed between [Pt(bipy)(NH₃)₂] ²⁺ and a series of dibenzo-3n-crown-n (n = 6–12) ethers as evidenced by ¹H NMR and UV-visible spectra in solution and by X-ray crystal structures (for n = 8 and 10) in the solid state. Mutual diamagnetic (ring current) shielding by the aromatic systems of host and guest results in dramatic upfield shifts of certain aromatic proton resonances on adduct formation, especially when n = 10 or 11. A broad charge-transfer absorption band at 350 nm attains its maximum intensity at n = 11 though stability constant measurements indicate optimum binding when n = 10. X-ray diffraction studies reveal that, when n = 8, only one of the crown ether benzo-rings interacts with the bipyridyl ligand in a charge-transfer sense. However, when n = 10, the macrocycle is sufficiently large and flexible to allow both benzo-rings to enter into stabilising interactions with the bipyridyl ligand such that the platinum complex is encapsulated by the host in a U-shaped cavity.     

The Solvation of the E. coli CheY Phosphorylation Site Mapped by XFMS

The Escherichia coli CheY protein belongs to a large bacterial response regulator superfamily. X-ray hydroxy radical foot-printing with mass spectroscopy (XFMS) has shown that allosteric activation of CheY by its motor target triggers a concerted internalization of aromatic sidechains. We reanalyzed the XFMS data to compare polar versus non-polar CheY residue positions. The polar residues around and including the 57D phosphorylated site had an elevated hydroxy radical reactivity. Bioinformatic measures revealed that a water-mediated hydrogen bond network connected this ring of residues with the central 57D. These residues solvated 57D to energetically stabilize the apo-CheY fold. The abundance of these reactive residues was reduced upon activation. This result was supported by the bioinformatics and consistent with the previously reported activation-induced increase in core hydrophobicity. It further illustrated XFMS detection of structural waters. Direct contacts between the ring residues and the phosphorylation site would stabilize the aspartyl phosphate. In addition, we report that the ring residue, 18R, is a constant central node in the 57D solvation network and that 18R non-polar substitutions determine CheY diversity as assessed by its evolutionary trace in bacteria with well-studied chemotaxis. These results showcase the importance of structured water dynamics for phosphorylation-mediated signal transduction.     

Performances of Fe-[Al, B]MFI catalysts in benzene hydroxylation with N2O The role of zeolite defects as host sites for highly active iron species

The catalytic performances of Fe-[Al, B]MFI zeolites in benzene hydroxylation with N₂O are studied as a function of the nature of the T atom (Al, B) and compared with the characterization of the nature of iron species determined by UV–vis diffuse reflectance spectroscopy. The results indicate that the mechanism for generation of iron species highly active in this reaction is the creation of hydroxyl nests in the zeolite framework due to framework to extra-framework migration of the T atom. The process is favoured when B is present as T atom, but occurs also in the case of Fe and Al as T atoms. Iron atoms migrate to these zeolite defect sites forming highly active Fe³⁺ isolated species in square pyramidal or highly distorted octahedral coordination. The process occurs during the catalyst pre-treatment as well as during the initial half an hour of time on stream, leading to an increase in the phenol productivity during this step. A model of these active sites is presented. The effect of the silica source in the preparation of these catalysts is also reported.