Excision-repair capacity of UV-irradiated strains of Escherichia coli and Streptococcus pneumoniae, estimated by plasmid recovery

Although the biological role of many bacterial repair genes is known, there is still an interest in evaluating the capacity of repair pyrimidine dimers in some strains. For this purpose, we have developed a rapid assay. Cells bearing a plasmid are UV irradiated and incubated to allow recovery. The plasmid DNA is extracted, purified and treated with UV endonuclease from Micrococcus luteus that specifically produces single strand breaks at the site of pyrimidine dimers. The amount of open circular and covalently closed circular forms of the plasmid DNA after treatment and post-incubation provides an estimate of the repair capability of the host strain. The wild type strain and the uvrA mutant of Escherichia coli were used to adjust the assay. The lexA mutant of E. coli has been tested and its repair capability is equivalent to that of wild-type strain. The assay has been extended to Streptococcus pneumoniae, which is naturally deficient in photoreactivation and SOS-like functions. This strain is efficient in the repair of pyrimidine dimers, formed after UV irradiation.     

Detection of two variant Vero toxin genes in Escherichia coli by capillary electrophoresis

Three methods [capillary electrophoresis (CE)-allele-specific PCR, CE-single-strand conformation polymorphism (SSCP) and CE-cleavase fragment length polymorphism (CFLP)] were developed in order to effect rapid and specific analysis of the vero toxin (VT)1 and VT2 genes of O157. The allele-specific polymerase chain reaction (PCR) method, which utilized specific duplex PCR with specific primers for VT1 and VT2, showed that VT1 and VT2 consisted of 174 and 128 bp, respectively. Subsequent CE analysis was carried out. Separation time was 4 min. SSCP, which utilized one primer set which reacted with both VT1 and VT2 in the PCR method, was followed by CE analysis of secondary structure of single-strand DNA. Two genes could be analyzed in approximately 18 min. CFLP, like SSCP, is a method for detecting mutation-induced changes in secondary structure of single-stranded DNA. The endonuclease cleavase I recognizes and cleaves the 5' side of hairpin loops in self-annealed single-strand DNA of PCR product 169 bp obtained from VT1 and VT2. The produced DNA fragments are analyzed by CE and the electrophelogram reveals a sequence-specific CFLP. Separation time was 6 min. These techniques are suitable for the detection and the identification of O157.     

Electrophoretic Behavior Study of Bacteria of Pseudomonas aeruginosa, Edwardsiella tarda and Enteropathogenic Escherichia coli by Capillary Electrophoresis with UV and Fluorescence Detection

Capillary electrophoresis approaches have been utilized for the study of bacteria under specific experimental conditions. The main objective within our research work was to study electrophoretic behaviors of Pseudomonas aeruginosa by means of capillary electrophoresis with UV and fluorescence detection. Edwardsiella tarda and Enteropathogenic escherichia coli were also included in the study. The results showed that proper pretreatment (vortexing or sonication) for each bacterial sample before injection was necessary to disperse the clusters of cells, which is helpful to observe the single peaks and better peak shape of bacteria during electrophoresis. Apart from this, it was found that ionic strength of buffer affected mobilities of Pseudomonas aeruginosa as a result of increasing of buffer concentration from 25 mM to 150 mM. Moreover, sharp and single peaks were still observed without significant increase of noise in the concentration range. Eventually, mixtures of bacteria were well separated under optimized separation conditions with UV and fluorescence detection. In the mean time, comparison of concentration sensitivities for Pseudomonas aeruginosa by UV and fluorescence detection was made. Blue light emitting diode induced fluorescence detection was found to be more sensitive (8.5-fold higher) than UV detection with home-made fluorescence detection system. Generally, proposed CE methods for the analysis of bacteria could be potentially valuable for the monitoring of bacteria contamination in real life.     

Antigenic specificity of Escherichia coli alkaline phosphatase studied with monoclonal antibodies: Immunological characterization of E. coli and Shigella strains

Monoclonal antibodies (MoAb) to the alkaline phosphatase of Escherichia coli were produced from spleen cells of BALB/c mice primed with purified alkaline phosphatase of E. coli and SP₂O/Ag-14 myeloma cells. Five stable clones were established. They all produced antibodies which reacted by enzyme-linked immunosorbent assay (ELISA) with alkaline phosphatase of all E. coli (25 strains) independently of their origin (drinking water, saline water, surface water, faecal or clinical origin), and with that of four Shigella species (7 strains) tested. Four of these MoAb gave a positive reaction with 52 % (MoAb 4G10), 73 % (MoAb 4F8, MoAb 4G6) and 89 % (MoAb 3C8) of 14 other bacterial species (30 strains) studied, while one (MoAb 2E5) did not react with alkaline phosphatase of these unrelated bacterial strains and thus appeared specific for E. coli and Shigella species. This MoAb was still detectable in ascitic fluids at 1/500,000 in ELISA, and detected all E. coli strains in an indirect immunofluorescence assay at 1/100. It could therefore be used as a reagent for routine detection of E. coli in drinking water, food or clinical specimens.     

Detection of Chlamydia trachomatis by in situ hybridization with sulphonated total DNA

In situ nucleic acid hybridization was applied to the detection of Chlamydia trachomatis on microscope slides by use of sulphonated total DNA as a probe. Visualization of labelled DNA was obtained using a commercial enzyme-linked monoclonal antibody. A mixture of paraformaldehyde and glutaraldehyde was found to be the best fixative. With high probe concentration (10 μg/ml), intracellular inclusions were detected as early as 8 h after inoculating the cell culture. Extracellular elementary bodies could also be detected. Five genital specimens were tested by in situ hybridization; the results were in agreement with those observed by culture.     

Probing roles of lipopolysaccharide, type 1 fimbria, and colanic acid in the attachment of Escherichia coli strains on inert surfaces

The roles of bacterial surface polymers in reversible (phase I) and irreversible (phase II) attachment (i.e., lipopolysaccharides (LPS), type 1 fimbria, and capsular colanic acid (CA)) were investigated in situ by combining fluorescence microscopy and atomic force microscopy. Fluorescence microscopy was used to evaluate the phase I attachment by counting the total number of cells on the substrata, and AFM was applied to image the phase II cells and measure the lateral detachment force to characterize phase II attachment. Also, by comparing the number of cells in phases I and II, the transformation ratio was calculated and used as an index to evaluate the roles of different polymers in the attachment process. Escherichia coli K-12 and its six mutants, which had different surface polymers in terms of LPS structures, CA contents, and type 1 fimbriae, were used as the test strains. Six different materials were applied as substrata, including glass, two metals (aluminum and stainless steel), and three plastics (polyvinyl chloride, polycarbonate, and polyethylene). The results indicated that LPS significantly enhanced phases I and II attachment as well as the transformation ratio from phase I to II. Like LPS, type 1 fimbriae largely increased the phase I attachment and the transformation ratio; however, they did not significantly influence the adhesion strength in phase II. CA had a negative effect on attachment in phases I and II by decreasing the adhered number of cells and the lateral detachment force, respectively, but had no influence on the transformation ratio.     

Detection of electronic and vibrational coherences in molecular systems by 2D electronic photon echo spectroscopy

Two-dimensional optical photon echo spectra are simulated for model systems which exhibit vibrational, electronic and a combination of electronic and vibrational coherent dynamics. The coherent motion manifests itself as periodic beatings of the spectrum cross-peak intensity with the population time. The intensity modulations are compared to evolution of the excited-state population and coordinate expectation value. The advantageous capabilities of the technique as well as possible difficulties in spectra interpretations are outlined. Possibilities for distinguishing electronic and vibrational coherences are discussed.     

Nanophotonic Device in Combination with Bacteriophages for Enhancing Detection Sensitivity of Escherichia coli in Simulated Wash Water

Among various approaches to control and monitor cross-contamination of fresh produce, a biosensor that can rapidly detect the presence of a specific bacterium in wash water or on fresh produce can be effective. This research demonstrates the development of a rapid biosensor based on a combination of a nanophotonic device and the bacteriophage T7 for the detection of Escherichia coli without the need for culturing or nucleic acid extraction. This biosensor platform is based on bacteriophage mediated specific lysis of target bacteria and release of β-galactosidase. The enzyme could be further detected by a nanophotonic device that amplifies the fluorescent signal, therefore allowing better sensitivity. Production of β-galactosidase is induced by isopropyl β-D-1-thiogalactopyranoside (IPTG) and the enzyme is then released by bacteriophage lysis, which is detected by the nanophotonic device using a fluorescent enzyme substrate resorufin β-D-galactopyranoside. Using this approach, the results demonstrated successful detection of 10 CFU mL^?1 of E. coli BL21 in simulated spinach wash water within 8?hours. Specificity of the assay was demonstrated with negative controls including Pseudomonas fluorescens and Listeria innocua.     

Numerical computations of molecular reactions in associated systems caused by the formation of fractal structures

The interaction between particles in a system containing fractal clusters has been computationally simulated. The fractal structure of the system has been demonstrated to determine the kinetic characteristics of particle interaction. If a system in an N-dimensional space (N = 2, 3, 4) contains fractal clusters with the fractal dimension D > N-1, the rate of interaction of a free particle with particles belonging to clusters depends on their concentration according to the power law. The exponent gamma of this power law formally corresponds to the kinetic order of the reaction with respect to the concentration of particles belonging to the clusters. Its value is determined by the free surface of the clusters and depends on its fractal dimension D. The results of simulation qualitatively agree with the data on high, non-integral orders of many liquid phase molecular reactions characterized by self-organization of the medium via weak intermolecular interactions, such as hydrogen bonds.     

Change in broth culture is associated with significant suppression of Escherichia coli death under high magnetic field

When Escherichia coli B was cultivated under an inhomogeneous magnetic field of 5.2-6.1 T, a significant 100,000-fold suppression of cell death was observed [Bioelectrochemistry 53 (2001) 149]. The limited magnetic field exposure for 12 h after logarithmic growth phase was sufficient to observe similar suppressive effects on cell death [Bioelectrochemistry 54 (2001) 101]. These results suggest some possible changes in either the medium or the cells during the magnetic field exposure. When the cell-free filtrate of the broth cultured under the magnetic field for 10 h and the cells of E. coli cultivated under the geomagnetic field for 30 h were mixed, and the mixture was subsequently cultivated under the geomagnetic field, the number of cells observed in the filtrate exposed to the high magnetic field was 20,000 times higher than that in the filtrate exposed to the geomagnetic field. When the cells cultivated under the magnetic field for 10 h and the cell-free filtrate of the broth culture exposed to the geomagnetic field were mixed, only a 50-fold difference in the number of cell between under the magnetic field and under the geomagnetic field was observed. This suggests that the filtrate of the broth culture exposed to the magnetic field is primarily responsible for the cell death suppression. It was also revealed that the small difference in pH of the filtrates of the broth culture between under the magnetic field and under the geomagnetic field was critical for the cell death suppression.     

Construction, expression, and characterization of recombinant hirudin in Escherichia coli

The mutant gene of HV2-K47 was obtained by polymerase chain reaction-directed mutagenesis and expressed in Escherichia coli. Many elements that could affect its expression level were compared. The product was purified to homogeneity via three chromatographic steps—ion exchange, gel filtration, and reverse phase chromatography—on the AKTA Explorer System. The antithrombin activity of HV2-K47 is much higher than that of recombinant HV2. Some properties and expression conditions were investigated systematically, which would be useful for further studies of hirudin and other small proteins.     

Action spectra for photoreactivation of mutation of prototrophy in strains of Escherichia coli possessing and lacking photoreactivating-enzyme activity

Abstract— The action spectrum and dose-rate dependence for photoreactivation of mutation to prototrophy in late-lag-phase cultures of Escherichia coli H³r30 (which lacks active photo-reactivating enzyme) are roughly similar to those for photoprotection from killing in other strains. It is suggested that photoreactivation of this mutation in H/r30 is an indirect effect, similar in mechanism to photoprotection. The action spectrum and dose-rate dependence for photoreactivation of mutation to prototrophy in late-lag-phase cultures of E. coli H³r30-R (which possesses active photoreactivating enzyme) are roughly similar to those for photoreactivation of killing in most other strains. It is suggested that photoreactivation of this mutation in H/r30-R is a direct effect at long wavelengths, but that there is an indirect component at short wavelengths. A quite different interpretation of these data is noted. Finally, it is found that, under the conditions of these experiments, indirect photoreactivation of killing in H/r30 and H/r30-R is weak or nonexistent.     

Detection of Escherichia coli O157:H7 bacteria by a combination of immunofluorescent staining and capillary electrophoresis

As the number of incidents of bacterial infections continues to rise around the globe, simpler, faster, and more sensitive diagnostic techniques are required to improve the safety of the food supply and to screen for potential bacterial infections in humans. We present here direct and indirect approaches for the detection of bacteria, which are based upon a combination of immunofluorescent staining and capillary electrophoresis. In the direct approach, Escherichia coli O157:H7 bacteria stained with fluorescein-tagged specific antibodies are detected by CE, while in the indirect approach fluorescein-tagged specific antibodies to E. coli are first captured by E. coli O157:H7 bacteria and then released and detected by CE. We have identified suitable bacteria staining and CE protocols, which involved a 10 mM Tris-borate-EDTA (TBE) buffer, 0.25 micro g antibody/1 million bacteria, and capillaries dynamically coated with poly-N-hydroxyethylacrylamide (polyDuramide). We have also successfully detected the presence of E. coli O157:H7 in contaminated meat. The total time required for analysis was 6-8 h, which is less than that realized in most commercial assays presently available.     

Characterization and comparison of metal accumulation in two Escherichia coli strains expressing either CopA or MntA, heavy metal-transporting bacterial P-type adenosine triphosphatases

MntA from Lactobacillus plantarum and copA from Enterococcus hirae both encode membrane proteins that are members of the P-type family of adenosine triphosphatases (ATPases). Both transporters act as metal importers to take up nutritionally required substrates; MntA translocates Mn(II) and CopA translocates Cu(I). Both ATPases can also translocate secondary substrates, Cd(II) and Ag(I), respectively. Although functionally and sequentially similar, these ATPases differ in several key residues and in their membrane topologies. The bioaccumulation properties of these two proteins were examined by coexpressing the transporters with overexpressed metallothionein in Escherichia coli cells, a system that has previously shown high levels of substrate-specific uptake. Both strains exhibited rapid metal accumulation, both saturated at around 50 μM metal, and both displayed temperature-sensitive uptake. However, the transporters responded differently when external conditions were varied; MntA displayed increased sensitivity to ionic strength, while CopA was more pH sensitive and more inhibited by chelating agents. The differences in accumulation are likely owing to structural differences in the transmembrane region of these two ATPases.     

Understanding the lid movements of LolA in Escherichia coli using molecular dynamics simulation and in silico point mutation

The Lol system in Escherichia coli is involved in localization of lipoproteins and hence is essential for growth of the organism. LolA is a periplasmic chaperone that binds to outer-membrane specific lipoproteins and transports them from inner membrane to outer membrane through LolB. The hydrophobic lipid-binding cavity of LolA consists of α-helices which act as a lid in regulating the transfer of lipoproteins from LolA to LolB. The current study aims to investigate the structural changes observed in LolA during the transition from open to closed conformation in the absence of lipoprotein. Molecular dynamics (MD) simulations were carried out for two LolA crystal structures; LolA(R43L), and in silico mutated MsL43R for a simulation time of 50 ns in water environment. We have performed an in silico point mutation of leucine to arginine in MsL43R to evaluate the importance of arginine to induce structural changes and impact the stability of protein structure. A complete dynamic analysis of open to closed conformation reveals the existence of two distinct levels; closing of lid and closing of entrance of hydrophobic cavity. Our analysis reveals that the structural flexibility of LolA is an important factor for its role as a periplasmic chaperone.     

Co-Production of Isoprene and Lactate by Engineered Escherichia coli in Microaerobic Conditions

Lactate and isoprene are two common monomers for the industrial production of polyesters and synthetic rubbers. The present study tested the co-production of D-lactate and isoprene by engineered Escherichia coli in microaerobic conditions. The deletion of alcohol dehydrogenase (adhE) and acetate kinase (ackA) genes, along with the supplementation with betaine, improved the co-production of lactate and isoprene from the substrates of glucose and mevalonate. In fed-batch studies, microaerobic fermentation significantly improved the isoprene concentration in fermentation outlet gas (average 0.021 g/L), compared with fermentation under aerobic conditions (average 0.0009 g/L). The final production of D-lactate and isoprene can reach 44.0 g/L and 3.2 g/L, respectively, through fed-batch microaerobic fermentation. Our study demonstrated a dual-phase production strategy in the co-production of isoprene (gas phase) and lactate (liquid phase). The increased concentration of gas-phase isoprene could benefit the downstream process and decrease the production cost to collect and purify the bio-isoprene from the fermentation outlet gas. The proposed microaerobic process can potentially be applied in the production of other volatile bioproducts to benefit the downstream purification process.     

Nucleotide-binding properties of adenylate kinase from Escherichia coli: A molecular dynamics study in aqueous and vacuum environments

Summary The complex of adenylate kinase with its transition-state inhibitor has been studied by molecular dynamics simulations in water and in vacuum environments with the GROMOS force field over a period of 300 ps. The adenylate kinase, a member of the nucleotide-binding protein family, was exemplarily chosen for the inspection of the nucleotide-binding properties in the active site. The ligand binding and the domain movements have been studied in detail over the simulation period and compared with the crystal structure. Secondary structure transitions and domain closures defined those parts of the structure which are involved in an induced-fit movement of the enzyme. The presence of more stable hydrogen bonds on the substrate side leads to the assumption that substrate binding is more specific than cosubstrate binding. Reliable results were achieved only if water was explicitly included in the simulation.Dedicated to Prof. Dr. J. Seydel on the oceasion of his 65th birthday.     

Volatile organic compounds generated by cultures of bacteria and viruses associated with respiratory infections

Respiratory infections (RI) can be viral or bacterial in origin. In either case, the invasion of the pathogen results in production and release of various volatile organic compounds (VOCs). The present study examines the VOCs released from cultures of five viruses (influenza A, influenza B, adenovirus, respiratory syncitial virus and parainfluenza 1 virus), three bacteria (Moraxella catarrhalis, Haemophilus influenzae and Legionella pneumophila) and Mycoplasma pneumoniae isolated colonies. Our results demonstrate the involvement of inflammation‐induced VOCs. Two significant VOCs were identified as associated with infectious bacterial activity, heptane and methylcyclohexane. These two VOCs have been linked in previous studies to oxidative stress effects. In order to distinguish between bacterial and viral positive cultures, we performed principal component analysis including peak identity (retention time) and VOC concentration (i.e. area under the peak) revealing 1‐hexanol and 1‐heptadecene to be good predictors. Copyright © 2015 John Wiley & Sons, Ltd.     

Detection of Escherichia coli using immunomagnetic separation and bacteriophage amplification coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

The application of whole cell analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has emerged as a valuable tool for rapidly identifying/detecting bacteria. This technique requires minimal sample preparation and is simple to perform, but is generally limited to purified samples of bacteria at concentrations greater than 1.0 x 10(6) cells/mL. In this paper, we describe a bacterial detection method that integrates immunomagnetic separation with bacteriophage amplification prior to MALDI-MS analysis. The developed method consists of three main stages: (1) isolation of a target bacterium by immunomagnetic separation; (2) infection of the immuno-captured bacterium with a lytic bacteriophage; and (3) assay of infected medium for bacteriophage progeny using MALDI-MS to produce a molecular weight signal for the virus capsid protein. With this technique, the presence of Escherichia coli in broth was determined in less then 2 h total analysis time at a concentration of approximately 5.0 x 10(4) cells/mL.