由吸附的缩氨酸诱导的细胞膜的形变

研究了由一系列相互平行的吸附在细胞膜上的缩氨酸引起的膜的弹性形变,以及膜对缩氨酸的包裹行为,得到膜的平衡方程,用它可以来处理大尺度的形变,弯曲能量、吸附能量和弹性形变的相互竞争导致膜对缩氨酸发生从不吸附到部分吸附乃至完全包裹的结构转变．在膜的形变很小的时候,可以得到系统能量的解析解。     

Surface Binding of Aflatoxin B1 by Lactic Acid Bacteria

Specific lactic acid bacterial strains remove toxins from liquid media by physical binding. The stability of the aflatoxin B₁ complexes formed with 12 bacterial strains in both viable and nonviable (heat- or acid-treated) forms was assessed by repetitive aqueous extraction. By the fifth extraction, up to 71% of the total aflatoxin B₁ remained bound. Nonviable bacteria retained the highest amount of aflatoxin B₁. Lactobacillus rhamnosus strain GG (ATCC 53103) and L. rhamnosus strain LC-705 (DSM 7061) removed aflatoxin B₁ from solution most efficiently and were selected for further study. The accessibility of bound aflatoxin B₁ to an antibody in an indirect competitive inhibition enzyme-linked immunosorbent assay suggests that surface components of these bacteria are involved in binding. Further evidence is the recovery of around 90% of the bound aflatoxin from the bacteria by solvent extraction. Autoclaving and sonication did not release any detectable aflatoxin B₁. Variation in temperature (4 to 37°C) and pH (2 to 10) did not have any significant effect on the amount of aflatoxin B₁ released. Binding of aflatoxin B₁ appears to be predominantly extracellular for viable and heat-treated bacteria. Acid treatment may permit intracellular binding. In all cases, binding is of a reversible nature, but the stability of the complexes formed depends on strain, treatment, and environmental conditions.     

Metabolism and Metal Binding by Surface-Colonizing Bacteria: Results of Microgradient Measurements

Short-term (65-h) bacterial colonization of 0.2-μm (pore size) filters submerged in water from Lake Charlotte, Nova Scotia, was characterized by a well-defined succession of cell types, in which small cocci gave way to larger, rod-shaped cells. This succession agrees with the concept of attachment as a strategy for survival, in which inactive cocci can attach to a surface and grow into larger, rod-shaped cells by using endogenous nutrients and the nutrients accumulated at the solid-liquid interface. Analyses of oxygen and CO₂ microgradients above colonized surfaces indicated that a peak of respiration accompanied the succession of rods from cocci. CO₂ fixation then became apparent as the rods began to bind manganese and iron to their surfaces. This means that survival by attachment may not be just the province of heterotrophs. It could also be a strategy adopted by metal-oxidizing chemoautotrophs. Long-term (34-day) colonization of similar filters indicated that, while a succession of attached cell types may indeed be a natural occurrence, other factors (such as the selective grazing of larger cells) tend to obscure the development of this succession.     

Positive Pressure Effect on Manganese Binding by Bacteria in Deep-Sea Hydrothermal Plumes

A positive pressure effect (1.4 to 3.3×) on the binding of Mn²⁺ by a natural population of bacteria in a deep-sea hydrothermal plume was discovered over the intermediate pressure range of 1 to 200 atm (1 to 200 bars; ca. 1.01 × 10² to 2.03 × 10⁴ kPa). The data suggest Mn²⁺ binding is functionally barophilic rather than simply barotolerant.     

Prediction of Protein Binding to DNA in the Presence of Water-Mediated Hydrogen Bonds

We extend our previous analysis of binding specificity of DNA-protein complexes to complexes containing water-mediated bridges. Inclusion of water bridges between phosphate and base, phosphate and sugar, as well as proteins and DNA, improves the prediction of specificity; six data sets studied in this paper yield correct predictions for all base pairs that have two or more hydrogen-bonds. Beside massive computation, our approach relies highly on experimental data. After deriving protein structures from DNA-protein complexes in which coordinates were established by X-ray diffraction techniques, we analysed all possible DNA sequences to which these proteins might bind, ranking them in terms of Lennard-Jones potential for the optimal docking configuration. Our prediction algorithm rests on the following assumptions: (1) specificity comes mainly from direct hydrogen bonding; (2) electrostatic forces stabilise DNA-protein complexes and contribute only weakly to specificity since they occur at the charged phosphate groups; (3) Van der Waals forces and electrostatic interactions between positively charged groups on the protein and phosphates on DNA can be neglected as they contribute primarily to the free energy of stabilisation as opposed to specificity.     

Calcium Binding and Disulfide Bonds Regulate the Stability of Secretagogin towards Thermal and Urea Denaturation

Secretagogin is a calcium-sensor protein with six EF-hands. It is widely expressed in neurons and neuro-endocrine cells of a broad range of vertebrates including mammals, fishes and amphibia. The protein plays a role in secretion and interacts with several vesicle-associated proteins. In this work, we have studied the contribution of calcium binding and disulfide-bond formation to the stability of the secretagogin structure towards thermal and urea denaturation. SDS-PAGE analysis of secretagogin in reducing and non-reducing conditions identified a tendency of the protein to form dimers in a redox-dependent manner. The denaturation of apo and Calcium-loaded secretagogin was studied by circular dichroism and fluorescence spectroscopy under conditions favoring monomer or dimer or a 1:1 monomer: dimer ratio. This analysis reveals significantly higher stability towards urea denaturation of Calcium-loaded secretagogin compared to the apo protein. The secondary and tertiary structure of the Calcium-loaded form is not completely denatured in the presence of 10 M urea. Reduced and Calcium-loaded secretagogin is found to refold reversibly after heating to 95°C, while both oxidized and reduced apo secretagogin is irreversibly denatured at this temperature. Thus, calcium binding greatly stabilizes the structure of secretagogin towards chemical and heat denaturation.     

Aggregative Behavior of Bacteria Isolated from Canine Dental Plaque

Interbacterial adhesion of bacteria isolated from canine dental plaque was assessed by performing a visual coaggregation assay. Using conditions mimicking those likely to be encountered in vivo, the entire cultivable plaque microbiota from a single dog was assessed, and eight (6.7%) unique coaggregation interactions were detected for 120 crosses. Transmission electron microscopy was used to visualize several of the bacteria in isolation and as coaggregates, which revealed surface structures that may be involved in adhesion and coaggregation. The results of this study indicate that the prevalence of coaggregating pairs of dental plaque bacteria in dogs is similar to the prevalence of coaggregating pairs of dental plaque bacteria reported in humans. In addition, genera found in both hosts generally exhibited similar coaggregation reactions; however, autoaggregation was found to be more common among oral bacteria isolated from dogs.     

Selectivity of Ferric Enterobactin Binding and Cooperativity of Transport in Gram-Negative Bacteria

The ligand-gated outer membrane porin FepA serves Escherichia coli as the receptor for the siderophore ferric enterobactin. We characterized the ability of seven analogs of enterobactin to supply iron via FepA by quantitatively measuring the binding and transport of their ⁵⁹Fe complexes. The experiments refuted the idea that chirality of the iron complex affects its recognition by FepA and demonstrated the necessity of an unsubstituted catecholate coordination center for binding to the outer membrane protein. Among the compounds we tested, only ferric enantioenterobactin, the synthetic, left-handed isomer of natural enterobactin, and ferric TRENCAM, which substitutes a tertiary amine for the macrocyclic lactone ring of ferric enterobactin but maintains an unsubstituted catecholate iron complex, were recognized by FepA (K_d ≈ 20 nM). Ferric complexes of other analogs (TRENCAM-3,2-HOPO; TREN-Me-3,2-HOPO; MeMEEtTAM; MeME-Me-3,2-HOPO; K₃MECAMS; agrobactin A) with alterations to the chelating groups and different net charge on the iron center neither adsorbed to nor transported through FepA. We also compared the binding and uptake of ferric enterobactin by homologs of FepA from Bordetella bronchisepticus, Pseudomonas aeruginosa, and Salmonella typhimurium in the native organisms and as plasmid-mediated clones expressed in E. coli. All the transport proteins bound ferric enterobactin with high affinity (K_d ≤ 100 nM) and transported it at comparable rates (≥50 pmol/min/10⁹ cells) in their own particular membrane environments. However, the FepA and IroN proteins of S. typhimurium failed to efficiently function in E. coli. For E. coli, S. typhimurium, and P. aeruginosa, the rate of ferric enterobactin uptake was a sigmoidal function of its concentration, indicating a cooperative transport reaction involving multiple interacting binding sites on FepA.     

Effect of Trichloroethylene on the Competitive Behavior of Toluene-Degrading Bacteria

The influence of trichloroethylene (TCE) on a mixed culture of four different toluene-degrading bacterial strains (Pseudomonas putida mt-2, P. putida F1, P. putida GJ31, and Burkholderia cepacia G4) was studied with a fed-batch culture. The strains were competing for toluene, which was added at a very low rate (31 nmol mg of cells [dry weight]⁻¹ h⁻¹). All four strains were maintained in the mixed culture at comparable numbers when TCE was absent. After the start of the addition of TCE, the viabilities of B. cepacia G4 and P. putida F1 and GJ31 decreased 50- to 1,000-fold in 1 month. These bacteria can degrade TCE, although at considerably different rates. P. putida mt-2, which did not degrade TCE, became the dominant organism. Kinetic analysis showed that the presence of TCE caused up to a ninefold reduction in the affinity for toluene of the three disappearing strains, indicating that inhibition of toluene degradation by TCE occurred. While P. putida mt-2 took over the culture, mutants of this strain which could no longer grow on p-xylene arose. Most of them had less or no meta-cleavage activity and were able to grow on toluene with a higher growth rate. The results indicate that cometabolic degradation of TCE has a negative effect on the maintenance and competitive behavior of toluene-utilizing organisms that transform TCE.     

Biosensor-Based Evaluation of Liposomal Behavior in the Target Binding Process

In this study we evaluated the quartz crystal microbalance (QCM) as a biosensor for a real-time investigation of liposomal binding, under dynamic flow conditions, onto target proteins immobilized at the sensor. The mass-sensitive frequency changes of quartz sensors allow for a quantification of the liposomal binding process. Furthermore, simultaneous damping analysis gives an insight into liposomal behavior, such as the degree of liposomal deformation or spreading at the target surface. In this study a series of liposomes was evaluated, differing in the kind and concentration of ligands interacting with appropriate target proteins. It became evident that an increase in homing device concentration accelerated deformation and flattening of liposomes, triggering a fusion process. Furthermore, liposomal deformation corresponded with the binding affinity of target molecules, comparing biotin/avidin with E-selectin/ligand interactions. Deformation could be emphasized using dioleoylphosphatidylethanolamine (DOPE) as a fusiogenic membrane component, while sterical stabilization by polyethylenglycol (PEG-PE) appeared in a low degree of deformation. Consequently, the online detection of liposomal target binding by QCM is an excellent facility to control and predict the liposomal behavior at the target site for increasing therapeutic potency.     

Binding of Streptavidin to Bacteria or Fungi and Its Applications in Detecting These Microbes

We have investigated the characteristics and utilities of streptavidin-binding to gram-negative and gram-positive bacteria and Candida spp. The pre-treatment of these microbes with chemical reagents such as CHCl₃, NaOH, and Tween 20 have allowed colorimetric visualization under light microscopy or quantitation on nitrocellulose membranes, using streptavidin/biotinylated alkaline phosphatase conjugates. Analysis of this binding was confirmed by western blot. These binding reactions were due to the specific interaction of streptavidin with biotinylated proteins present in the microbes. Competition assays with free biotin or inhibition by an antibiotin antibody confirmed binding to these proteins. With knowledge of these strongly specific interactions, we attempted to reveal the biotinylated proteins within these microbes using clinical specimens. Using phagocyte-smears from blood, urine, and ascites, these intracellular microbes were easily detected by light microscopy. One of the septic blood samples stained by our technique revealed semi-digested microbial signals despite the absence of a signal with routine staining. This detection system, which combines streptavidin as a probe and biotinylated proteins as a microbial marker, is useful in staining for intracellular bacteria or fungi (e.g., microbial infections in phagocyte-smears).     

Inter-specific Interactions Between Carbon-limited Soil Bacteria Affect Behavior and Gene Expression

Recent publications indicate that inter-specific interactions between soil bacteria may strongly affect the behavior of the strains involved, e.g., by increased production of antibiotics or extracellular enzymes. This may point at an enhanced competitive ability due to inter-specific triggering of gene expression. However, it is not known if such inter-specific interactions also occur during competition for carbon which is the normal situation in soil. Here, we report on competitive interactions between two taxonomically non-related bacterial strains, Pseudomonas sp. A21 and Pedobacter sp. V48, that were isolated from a dune soil. The strains showed strong effects on each other’s behavior and gene expression patterns when growing together under carbon-limited conditions on agar. The most pronounced observed visual changes in mixed cultures as compared to monocultures were (1) strong inhibition of a bioindicator fungus, suggesting the production of a broad-spectrum antibiotic, and (2) the occurrence of gliding-like movement of Pedobacter cells. Two independent techniques, namely random arbitrary primed-PCR (RAP-PCR) and suppressive subtractive hybridization (SSH), identified in total 24 genes that had higher expression in mixed cultures compared to monocultures. Microbial interactions were clearly bidirectional, as differentially expressed genes were detected for both bacteria in mixed cultures. Sequence analysis of the differentially expressed genes indicated that several of them were most related to genes involved in motility and chemotaxis, secondary metabolite production and two-component signal transduction systems. The gene expression patterns suggest an interference competition strategy by the Pseudomonas strain and an escape/explorative strategy by the Pedobacter strain during confrontation with each other. Our results show that the bacterial strains can distinguish between intra- and inter-specific carbon competition.     

Tuning of Thioredoxin Redox Properties by Intramolecular Hydrogen Bonds

Thioredoxin-like proteins contain a characteristic C-x-x-C active site motif and are involved in a large number of biological processes ranging from electron transfer, cellular redox level maintenance, and regulation of cellular processes. The mechanism for deprotonation of the buried C-terminal active site cysteine in thioredoxin, necessary for dissociation of the mixed-disulfide intermediate that occurs under thiol/disulfide mediated electron transfer, is not well understood for all thioredoxin superfamily members. Here we have characterized a 8.7 kD thioredoxin (BC3987) from Bacillus cereus that unlike the typical thioredoxin appears to use the conserved Thr8 side chain near the unusual C-P-P-C active site to increase enzymatic activity by forming a hydrogen bond to the buried cysteine. Our hypothesis is based on biochemical assays and thiolate pK_a titrations where the wild type and T8A mutant are compared, phylogenetic analysis of related thioredoxins, and QM/MM calculations with the BC3987 crystal structure as a precursor for modeling of reduced active sites. We suggest that our model applies to other thioredoxin subclasses with similar active site arrangements.     

Injury of Bacteria by Sanitizers

Injury of test cultures was quantitated by differences in colony counts obtained with a complete medium and those obtained on conventional selective media. Staphylococcus aureus, Streptococcus faecalis, and several strains of Escherichia coli were injured when exposed to the quaternary ammonium compound methylalkyltrimethyl ammonium chloride. Representative hypochlorite sanitizers also caused injury of E. coli ML30. Sanitizer concentration appeared to be the main factor in the cause of death and injury, a higher concentration being needed to cause death. Increases in temperature did not result in substantial increases in injury; however, the lethal effect was greater at higher temperatures. Varying the cell concentration from 10(7) to 10(9) cells per ml did not change the fraction of cell population killed or injured. The inability or failure of common selective media to detect injured bacteria in food could have serious public health consequences.     

Selection of Peptides for Lipopolysaccharide Binding on to Epoxy Beads and Selective Detection of Gram-negative Bacteria

Lipopolysaccharide (LPS)-binding peptides were enriched by using epoxy beads as a novel support to immobilize LPS for a phage displayed peptide library screening. The sequence of Phe-Ala-Pro-Trp (FAPW) was the most significant consensus motif of 10 selected clones, and Pro-Phe (PF) was the key dipeptide for binding at the apex of the loop to form a characteristic structure of CXXPFXXXC. Moreover, AWLPWAK, one of the highly conserved heptamer peptides, could detect specifically Gram-negative bacteria via a whole cell binding test at 10⁶ cells ml⁻¹. Received 12 July 2005; Revisions requested 1 August 2005 and 26 September 2005; Revisions received 12 September 2005 and 25 October 2005; Accepted 1 November 2005     

Cleavage of Disulfide Bonds of Wheat Glutenin by Mercuric Chloride

The dissociation of wheat glutenin into subunits was observed by treatment with a small amount of mercuric chloride under moderate conditions, suggesting that the cleavage of inter-polypeptide chain disulfide bonds in the glutenin might occur. The dissociation into the subunits was examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The electrophoretic patterns of the glutenin treated with mercuric chloride were essentially similar to those of the glutenin treated with 2-mercaptoethanol. Silver nitrate also had the same effects as mercuric chloride, and p-chloromercuribenzoate and N-ethylmaleimide showed no effect on the dissociation of the glutenin. Complete dissociation was achieved when the glutenin solution containing 0.5% SDS and 0.01 m phosphate buffer (pH 7.0) was incubated with 10^?3 m mercuric chloride (about four moles per mole of disulfide groups) at 30°C for 20 hr. Partial dissociation was also observed after 30 min incubation. Increasing temperature and SDS concentration promoted the rate of the dissociation of the glutenin by mercuric chloride.     

Study of Sugar Binding to the Sucrose-specific ScrY Channel of Enteric Bacteria Using Current Noise Analysis

CACO-2 BBE was used to determine the response of a gastrointestinal epithelium to tumor necrosis factor-α (TNF). Incubation of CACO-2 BBE with TNF did not produce any effect on transepithelial resistance (TER) within the first 6 hr but resulted in a 40–50% reduction in TER and a 30% decrease in I _sc (short circuit current) relative to time-matched control at 24 hr. The decrease in TER was sustained up to 1 week following treatment with TNF and was not associated with a significant increase in the transepithelial flux of [¹⁴C]-d-mannitol or the penetration of ruthenium red into the lateral intercellular space. Dilution potential and transepithelial ²²Na⁺ flux studies demonstrated that TNF-treatment of CACO-2 BBE cell sheets increased the paracellular permeability of the epithelium to Na⁺ and Cl⁻. The increased transepithelial permeability did not associate with an increase in the incidence of apoptosis. However, there was a TNF-dependent increase in [³H]-thymidine labeling that was not accompanied by a change in DNA content of the cell sheet. The increase in transepithelial permeability was concluded to be across the tight junction because: (i) 1 mm apical amiloride reduced the basolateral to apical flux of ²²Na⁺, and (ii) dilution potential studies revealed a bidirectionally increased permeability to both Na⁺ and Cl⁻. These data suggest that the increase in transepithelial permeability across TNF-treated CACO-2 BBE cell sheets arises from an alteration in the charge selectivity of the paracellular conductive pathway that is not accompanied by a change in its size selectivity. Received: 4 March 1997/Revised: 3 November 1997