Miscibility behavior and nanostructure of monolayers of the main phospholipids of Escherichia coli inner membrane

We report a thermodynamic study of the effect of calcium on the mixing properties at the air-water interface of two phospholipids that mimic the inner membrane of Escherichia coli: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol. In this study, pure POPE and POPG monolayers and three mixed monolayers, χ(POPE) = 0.25, 0.5, and 0.75, were analyzed. We show that for χ(POPE) = 0.75, the values of the Gibbs energy of mixing were negative, which implies attractive interactions. We used atomic force microscopy to study the structural properties of Langmuir-Blodgett monolayers that were transferred onto mica substrate at lateral surface pressures of 25 and 30 mN m(-1). The topographic images of pure POPE and POPG monolayers exhibited two domains of differing size and morphology, showing a step height difference within the range expected for liquid-condensed and liquid-expanded phases. The images captured for χ(POPE) = 0.25 were featureless, and for χ(POPE) = 0.5 small microdomains were observed. The composition that mimics quantitatively the proportions found in the inner membrane of E. coli , χ(POPE) = 0.75, showed large liquid condensed domains in the liquid expanded phase. The extension of each domain was quantitatively analyzed. Because calcium is used in the formation of supported bilayers of negatively charged phospholipids, the possible influence of the nanostructure of the apical on the distal monolayer is discussed.     

Photodynamic action of methylene blue: repair and mutation in Escherichia coli

The effects of the photodynamic action of methylene blue (MB-PDA) on strains of Escherichia coli were investigated to determine whether the dye could be used in photodynamic therapy (PDT). Using the method of alkaline sucrose gradient sedimentation, it was shown that in darkness MB induces a type of prelesion in DNA that transforms into single-strand breaks in alkaline conditions, provided that the dye is present during the processing of the gradient. This prelesion is completely reversible if the cells are washed immediately to remove the dye. However, after illumination with white light, the prelesions become "fixed" stable lesions, irreversible even after successive washings. The lethal damage induced by MB-PDA in E. coli can be repaired by the excision-repair system (about 30%) and by the recA-dependent repair system (about 70%). Polymerase I enzyme participates actively in the repair of the damage. MB-PDA is a weak mutagen and the induction of mutations by this treatment is restricted to high survival rates. Moreover, MB-PDA does not induce the SOS system (an inducible repair system dependent of the recA and loxA genes products), as measured by Weigle reactivation. However, it seems that this treatment can impair the repair systems in E. coli.     

Effect of Glucose on Photodynamic Action of Methylene Blue in Escherichia coli Cells

The results of this work show that the resistance of Escherichia coli cells to the photodynamic action of methylene blue is increased by the addition of glucose to the media in which they are grown. It is postulated that the increased resistance may be due to lowered retention of the dye by cells grown in the presence of glucose, leading to the diminution in DNA damage revealed in the alkaline sucrose gradients. The role of cyclic adenosine-monophosphate in the protective action of glucose is discussed.     

Shotgun lipidomics for candidate biomarkers of urinary phospholipids in prostate cancer

Qualitative and quantitative profiling of six different categories of urinary phospholipids (PLs) from patients with prostate cancer was performed to develop an analytical method for the discovery of candidate biomarkers by shotgun lipidomics method. Using nanoflow liquid chromatography–electrospray ionization–tandem mass spectrometry, we identified the molecular structures of a total of 70 PL molecules (21 phosphatidylcholines (PCs), 11 phosphatidylethanolamines (PEs), 17 phosphatidylserines (PSs), 11 phosphatidylinositols (PIs), seven phosphatidic acids, and three phosphatidylglycerols) from urine samples of healthy controls and prostate cancer patients by data-dependent collision-induced dissociation. Identified molecules were quantitatively examined by comparing the MS peak areas. From statistical analyses, one PC, one PE, six PSs, and two PIs among the PL species showed significant differences between controls and cancer patients (p < 0.05, Student’s t test), with concentration changes of more than threefold. Cluster analysis of both control and patient groups showed that 18:0/18:1-PS and 16:0/22:6-PS were 99% similar in upregulation and that the two PSs (18:1/18:0, 18:0/20:5) with two PIs (18:0/18:1 and 16:1/20:2) showed similar (>95%) downregulation. The total amount of each PL group was compared among prostate cancer patients according to the Gleason scale as larger or smaller than 6. It proposes that the current study can be utilized to sort out possible diagnostic biomarkers of prostate cancer.     

Inner membrane lipids of Escherichia coli form domains

In prokaryotic cells, the hypothesis of the existence of lipid domains was considered. In order to test this hypothesis and study the organization of lipids in the inner membrane of Escherichia coli, we elaborated Langmuir films mimicking the inner leaflet of this membrane by considering lipids extracted from the inner membrane of E coli by Folch protocol.

Lipid monolayers were elaborated by using these extracts (Langmuir technique); the organization of the resulting films was studied at the air–water interface by Brewster angle microscopy and after transfer onto muscovite by atomic force microscopy. The existence of domains was demonstrated for different interfacial pressures of biological interest, and their stability was studied.     

Harvest time effects on membrane cake resistance of Escherichia coli broth

The nature of the ultrafiltration resistance of Escherichia coli broth harvested at different culture times was investigated. E. coli broth was harvested at the point of glucose depletion (0–h aged broth) and at 12 h after that (12 h aged broth). The filtration resistance of whole broth nearly doubled during the 12 h aging. In the case of the 0 h aged broth, the dominant resistance was from the deposited cells. However, for the 12 h aged broth, an interaction between cells and soluble broth components determined the filtration resistance. If either were missing, the resistance increase was markedly less. The cause of the interaction appeared to be a change in the cell (surface) during the aging, leading to binding with some broth component(s) that increased the resistance. The increased resistance occurred both when broth components were present during cake formation and when exposure came after cake deposition from washed cells. The interacting soluble component permeated a 0.16 μm membrane and was retained by a 300 kDa MWCO membrane. Since DNA would show such permeation and DNA added to aged, washed cells increased resistance in a similar fashion as occurred with aged broth, it is likely that DNA released from cells contributes to the interaction with aged cells.     

Redox sensing by Escherichia coli: effects of copper ions as oxidizers on proton-coupled membrane transport

Escherichia coli is able to grow under anaerobic fermentation conditions upon a decrease in redox potential (E(h)). Indeed, upon a transition of E. coli MC4100 wild-type culture to stationary growth phase a decrease in E(h) from the positive values ( approximately +100 mV) to the negative ones ( approximately -520 mV) was observed, the acidification of the medium and the H(2) production were obtained. An oxidizer, copper ions (Cu(2+)) affected a bacterial growth in a concentration-dependent manner (of 0.1 mM to 10 mM) increasing latent (lag) growth phase duration, delaying logarithmic (log) growth phase and decreasing specific growth rate. Acidification of the medium and the N,N'-dicyclohexylcarbodiimide (DCCD)- and azide-sensitive proton-potassium exchange by bacteria were inhibited, H(2) production upon growth and under assays disappeared with Cu(2+) (0.1 mM). These effects were observed with hycE but not hyfR and hyc(A-H) mutants and under aerobic conditions. Cu(2+) also increased membrane proton conductance. Copper ions are suggested to affect directly the F(0)F(1)-ATPase associated with potassium uptake transport system and/or formate hydrogenlyase composed with hydrogenase 4. A role of the F(0)F(1)-ATPase in redox sensing under fermentation is proposed.     

Photodynamic studies and photoinactivation of Escherichia coli using meso-substituted cationic porphyrin derivatives with asymmetric charge distribution

The photodynamic activities of novel asymmetrically meso-substituted cationic porphyrins, 5,10-di(4-methylphenyl)-15,20-di(4-trimethylammoniumphenyl)porphyrin iodide 1 and 5-(4-trifluorophenyl)-10,15,20-tris(4-trimethylammoniumphenyl)porphyrin iodide 2 and its metal complex with Pd(II) 3, have been investigated in both homogeneous medium bearing photooxidizable substrates and in vitro on a typical gram-negative bacterium Escherichia coli. The amphiphilic character of porphyrin 2 was increased by the presence of a high-lipophilic trifluoromethyl group and its photophysical properties changed by forming a complex with Pd(II). Absorption and fluorescence spectroscopic studies were compared in different media. Fluorescence quantum yields (phi(F)) of 0.16 for 1 in tetrahydrofuran and 0.08 for 2 in N, N-dimethylformamide (DMF) were calculated, whereas no significant emission was detected for Pd(II) porphyrin 3. The singlet molecular oxygen, O(2)((1)Delta(g)), production was evaluated using 9,10-dimethylanthracene in DMF yielding relative values of 1, 0.55 and 0.47 for porphyrins 3, 2 and 1, respectively. A faster decomposition of l-tryptophan was obtained using Pd(II) porphyrin 3 as sensitizer with respect to the free-base porphyrins 1 and 2. In biological medium, the behavior of cationic porphyrins 1-3 were compared with that of 5-(4-carboxyphenyl)-10,15,20-tris(4-methylphenyl)porphyrin 4, which was used as a noncationic sensitizer. These porphyrins are rapidly bound to E. coli cells in 5 min and the amount of cell-bound sensitizer is not appreciably changed incubating the cultures for longer times. The recovered porphyrin 2 after one washing step reaches a value of approximately 2.9 nmol/10(6) cells and this amount remains high even after three washes, indicating that this sensitizer is tightly bound to cells. Photosensitized inactivation of E. coli was analyzed using cells without and with one washing step. In both cases, a higher photoinactivation of cells was found for tricationic porphyrin 2 and 3, causing a approximately 5.5 log (99.999%) decrease of cell survival, when treated with 10 microM of sensitizer. Under these conditions, a lower effect was found for porphyrin 1 (approximately 4 log) whereas sensitizer 4 did not produce appreciable photodamage. The results were also confirmed by growth delay experiments. These studies show that the amphiphilic tricationic porphyrin 2 and 3 bearing a trifluoromethyl group can be a promising model for phototherapeutic agents with potential applications in inactivation of bacteria by photodynamic therapy.     

Structure of C-1 substituted glycopyranosyl azides: new insights based on CD measurements

CD spectra have been recorded for a series of peracetylated d-glycopyranosyl azides (d-gluco, d-galacto, d-xylo, d-arabino configuration) substituted at the anomeric position by various groups: amido, azido, cyano, ethoxy, methoxy. Application of the azide octant rule for the interpretation of the sign for the long-wavelength azide band allowed conformation of the azido group in each mono azido derivative investigated to be established. In each 1-cyano derivative, the azido group was in a gauche-like arrangement with respect to the C-1–O^ring bond, which is considered as a manifestation of the exo-anomeric effect of the azido group. For the 1-alkoxy derivatives, an antiparallel orientation of the azido group with respect to the C-1–O^ring bond was found in solution by CD measurement analysis, as already observed for methoxyazide 5 in the solid state. For azidoamide derivatives, intramolecularly (N–H–N^x_azide) H-bonded conformers are believed to prevail in methanol, in contrast to the situation in DMSO.     

Rapid identification of diarrheagenic Escherichia coli based on barcoded magnetic bead hybridization

Diarrhea, as a global public health problem, causes a large number of infections and deaths every year. Although Escherichia coli (E. coli) is one of the normal flora microorganisms in the human intestinal tract, it has five pathogenic bacteria types that can cause human diarrhea, known as diarrheagenic E. coli. When people are infected, rapid and accurate diagnosis, along with timely treatment, are especially important. Here, we introduce a new method to identify and analyze a large number of pathogenic strains in E. coli by multiplex PCR and barcoded magnetic bead hybridization. Results show that the detection sensitivities of enterohemorrhagic E. coli, enterotoxigenic E. coli, enteropathogenic E. coli, enteroinvasive E. coli and enteroaggregative E. coli were 1.3 × 10³ CFU/mL, 2 × 10⁴ CFU/mL, 4 × 10⁴ CFU/mL, 7.2 × 10⁴ CFU/mL and 1.7 CFU/mL respectively. This method has strong specificity and high sensitivity and detects multiple target sequences in one experiment. Compared with other methods, BMB array has great application potential.     

Nanostructured zirconium oxide based genosensor for Escherichia coli detection

A deoxyribonucleic acid (DNA) biosensor has been fabricated via immobilization of 17 base terminal single stranded DNA (ssDNA) identified from the 16s rRNA coding region of Escherichia coli onto sol–gel derived nanostructured zirconium oxide (NanoZrO₂) film. An oligonucleotide probe with a terminal 5′-phosphate group has been attached to the surface of the electrode via affinity of NanoZrO₂ for phosphate. The results of hybridization studies carried out with the complementary, non-complementary and genomic DNA reveal that ssDNA/NanoZrO₂/ITO bioelectrode has a high selectivity and sensitivity towards hybridization detection with limits of 10^?6–10⁶ pM of complementary DNA.     

Overexpression and characterization of the Rhodobacter sphaeroides PufX membrane protein in Escherichia coli

Heterologous expression of the PufX membrane protein from purple photosynthetic bacterium Rhodobacter sphaeroides was attempted by using Escherichia (E.) coli cells. The PufX was overexpressed as a recombinant protein with a histidine tag added to the carboxyl terminus, and can be extracted from the cell membrane by various detergents. Circular dichroism measurements showed that the expressed PufX protein had alpha-helix contents of 29% in organic solvents and 22-26% in 0.8-2.0% (w/v) n-octyl beta-D-glucopyranoside solutions, suggesting that the PufX contains a substantial alpha-helical region composed of 18-22 amino acids. The PufX expressed in E. coli was examined by reconstitution experiments with LH1 alpha- and beta-polypeptides and bacteriochlorophyll a. It was shown that the PufX inhibited not only the reconstitution of the LH1 complex, but also the formation of the B820 subunit type complex at high concentrations, indicating that the expressed PufX is biologically active. Large-scale expression of the functional PufX membrane protein provides sufficient quantity for further biophysical and structural analyses of its biological function, and adds another example for producing highly hydrophobic integral membrane proteins using the E. coli expression system.     

Perchlorate-selective membrane electrode based on a new complex of uranil

A potentiometric ion-selective electrode based on new compound, as a carrier, has been successfully developed for detection of perchlorate anion in aqueous solution. Within the perchlorate ion concentration range 1.0×10^–6 to 1.0 mol L^–1 the electrode had a linear response with a Nernstian slope of 60.6±1.0 mV per decade . The limit of detection as determined from the intersection of the extrapolated linear segments of the calibration plot was 8.0×10^–7 mol L^–1. The proposed electrode has fairly a good discriminating ability towards ClO₄^– ion in comparison to other anions. The sensor has a response time of 10 s and can be used for at least 2 months without substantial divergence in potential. It was successfully applied to direct determination of perchlorate in urine and water.     

A novel mediatorless microbial fuel cell based on direct biocatalysis of Escherichia coli

A mediatorless microbial fuel cell based on the direct biocatalysis of Escherichia coli shows significantly enhanced performance by using bacteria electrochemically-evolved in fuel cell environments through a natural selection process and a carbon/PTFE composite anode with an optimized PTFE content.