Microbial biosensor array with transport mutants of Escherichia coli K12 for the simultaneous determination of mono-and disaccharides

An automated flow-injection system with an integrated biosensor array using bacterial cells for the selective and simultaneous determination various mono- and disaccharides is described. The selectivity of the individually addressable sensors of the array was achieved by the combination of the metabolic response, measured as the O(2) consumption, of bacterial mutants of Escherichia coli K12 lacking different transport systems for individual carbohydrates. Kappa-carrageenan was used as immobilization matrix for entrapment of the bacterial cells in front of 6 individually addressable working electrodes of a screen-printed sensor array. The local consumption of molecular oxygen caused by the metabolic activity of the immobilized cells was amperometrically determined at the underlying screen-printed gold electrodes at a working potential of -600 mV vs. Ag/AgCl. Addition of mono- or disaccharides for which functional transport systems exist in the used transport mutant strains of E. coli K12 leads to an enhanced metabolic activity of the immobilized bacterial cells and to a concomitant depletion of oxygen at the electrode. Parallel determination of fructose, glucose, and sucrose was performed demonstrating the high selectivity of the proposed analytical system.     

Rapid electrochemical detection of polyaniline-labeled Escherichia coli O157:H7

There is a high demand for rapid, sensitive, and field-ready detection methods for Escherichia coli O157:H7, a highly infectious and potentially fatal food and water borne pathogen. In this study, E. coli O157:H7 cells are isolated via immunomagnetic separation (IMS) and labeled with biofunctionalized electroactive polyaniline (immuno-PANI). Labeled cell complexes are deposited onto a disposable screen-printed carbon electrode (SPCE) sensor and pulled to the electrode surface by an external magnetic field, to amplify the electrochemical signal generated by the polyaniline. Cyclic voltammetry is used to detect polyaniline and signal magnitude indicates the presence or absence of E. coli O157:H7. As few as 7CFU of E. coli O157:H7 (corresponding to an original concentration of 70 CFU/ml) were successfully detected on the SPCE sensor. The assay requires 70 min from sampling to detection, giving it a major advantage over standard culture methods in applications requiring high-throughput screening of samples and rapid results. The method can be performed with portable, handheld instrumentation and no biological modification of the sensor surface is required. Potential applications include field-based pathogen detection for food and water safety, environmental monitoring, healthcare, and biodefense.     

Recognitory bacterial surface lectins which mediate its mannose-specific adherence to eukaryotic cells

Cell surface protein were found to play a role in the sugar-specific molecular mechanism by which bacteria adhere to mammalian cells. We have demonstrated that at least three different types of lectin-like proteins mediate the mannose-sensitive adherence of gram negative bacteria to epithelial cells. One group of such lectins was shown in our study to be associated with the bacterial flagellum. Flagella isolated from Escherichia coli 7343 and Serratia marcescens 8347 exhibited mannose-sensitive agglutination of yeast cells; however, the flagella of the two bacteria differ in the molecular structure of their protein subunits. Another class of lectins comprises the bacterial fimbriae (also known as type 1 pili), which were previously shown to facilitate the mannose-sensitive adherence of various bacteria to mammalian cells. Fimbriae isolated from E. coli 346 were reversibly dissociated by saturated guanidine hydrochloride to their protein subunits. The dissociated subunits retained in part their mannose-binding ability, and were reassembled into fimbriae-like structures by removal of the denaturant under specific conditions. Mannose-sensitive yeast agglutinating activity of E. coli 2699, as well as of its isolated outer membranes devoid of fimbriae or flagella, was abolished by pretreatment with trypsin. It is therefore believed that the mannose-sensitive adherence of these bacteria is mediated also by lectin-like proteins associated directly with the outer membrane.     

Effects of growth phase and boiling of enteropathogenic Escherichia coli strains on their interaction with Pseudomonas aeruginosa lectins

Escherichia coli strains from' serotypes O86, 0128 and O111 varied in their reactivity with Pseudomonas aeruginose lectins (PA-I with D-galactose specificity and PA-II which binds L-fucose, D-mannose, L-galactose and D-fructose). Generally, cells of O86 strains were agglutinated by PA-I, but not by PA-II, and those of O128 serotype were agglutinated by PA-II, and not by PA-I. Adsorption tests showed that cells of E. coli O86 strains adsorb PA-I to a greater extent than PA-II, while most E. coli O128 strains adsorbed higher amounts of PA-II. Cells of E. coli O111B4 which were not agglutinated by either Pseudomonas lectin could still adsorb both. Boiling of O86 and O128 cells frequently enhanced their agglutinability as well as their lectin adsorption capacity. The agglutinability enhancement was somewhat more prominent in boiled stationary phase cells than in log phase cells probably due to late synthesis of the O antigen components concomitantly with the heat-sensitive components (K antigens) which masked them. PA-I agglutinating activity was inhibited by the lipopolysaccharide (LPS) extracted from E. coli O86 cells, while PA-II was inhibited by the LPS extracted from E. coli O128 cells. These findings indicate that the receptors to the Pseudomonas lectins probably reside in the terminal part of the O-specific-polysaccharide of the LPSs of these bacteria.     

Damage of Escherichia coli cells by t-butylhydroperoxide involves the respiratory chain but is independent of the presence of oxygen

The action of t-butylhydroperoxide (tBOOH) on Escherichia coli cells has been studied as a model system for organic peroxide toxicity. Exposure of E. coli cells to tBOOH led to progressive and irreversible impairment of the respiratory function, an effect which was dependent on the availability of substrate. The effect of tBOOH on growth of E. coli with different carbon sources and alternative terminal electron acceptors was investigated. It was found that the sensitivity of E. coli to tBOOH under diverse growth conditions implicating a functional respiratory chain was greater than when the bacterium grew by fermentation. Also the mutant E. coli SASX76, which requires exogenous 5-aminolevulinic acid to synthesize the cytochromes, was more resistant to tBOOH when lacking a functional respiratory chain. These data point to the respiratory chain as a major target in the in vivo action of tBOOH. Experiments with isolated membranes also showed a tBOOH-induced damage of the respiratory chain monitored by impairment of the NADH oxidase. The effect of tBOOH was produced even under anaerobiosis, indicating that development of cell damage was independent of oxygen and, therefore, that neither oxygen-derived radicals nor lipid peroxidation were involved.     

Rapid biosensor for detection of antibiotic-selective growth of Escherichia coli

A rapid biosensor for the detection of bacterial growth was developed using micromechanical oscillators coated in common nutritive layers. The change in resonance frequency as a function of the increasing mass on a cantilever array forms the basis of the detection scheme. The calculated mass sensitivity according to the mechanical properties of the cantilever sensor is approximately 50 pg/Hz; this mass corresponds to an approximate sensitivity of approximately 100 Escherichia coli cells. The sensor is able to detect active growth of E. coli cells within 1 h. The starting number of E. coli cells initially attached to the sensor cantilever was, on average, approximately 1,000 cells. Furthermore, this method allows the detection of selective growth of E. coli within only 2 h by adding antibiotics to the nutritive layers. The growth of E. coli was confirmed by scanning electron microscopy. This new sensing method for the detection of selective bacterial growth allows future applications in, e.g., rapid antibiotic susceptibility testing.     

Interaction of certain RNAases and mannose-specific lectins

While defining and elaborating the approaches to examination of the interaction between C-mannosylated tryptophan, recently discovered in the laboratory of Dr. Jan Hofsteenge, and Man/Glc specific lectins the unexpected results were obtained. Some animal origin mannosyl-containing RNases (as was expected) as well as analogous but nonglycosylated recombinant proteins expressed in E. coli (a negative control) were recognized by the mentioned lectins. Protein-protein interactions between lectins and expressed in E. coli nonglycosylated RNases are supposed and require further investigations.     

The phototoxicity of phenothiazinium-based photosensitizers to bacterial membranes

The ability of phenothiazinium-based photosensitizers to induce photodamage to Escherichia coli membranes is investigated. Phenothiazinium-based photosensitizers were found to be somewhat lipophilic (log P>0.7) and to induce surface-pressure changes (3-12 mN m(-1)) in lipid monolayers mimetic of bacterial membranes, implying that these molecules are able to penetrate biological membranes. Under dark and light conditions (3.15 J cm(-1) for 30 min), phenothiazinium-based photosensitizers were incubated with E. coli cells. These cells showed levels of dark bacteriolysis that ranged between 6% and 13%, with light conditions leading to no significant increase in these levels. Gas chromatography-based analyses showed such incubations to produce no significant changes in the levels of C(16) and C(18) fatty acid chain saturation found in E. coli whole lipid-extracts. It is concluded that the phenothiazinium-based photosensitizers studied may not use E. coli membranes as their primary photodynamic target, but may inflict photodamage on cytoplasmic targets, possibly DNA.     

Escherichia coli and its application in a mediated amperometric glucose sensor

Escherichia coli cells, which contain apo-glucose dehydrogenase, were used in constructing a mediated amperometric glucose sensor. The E. coli modified glucose sensor, which was prepared by immobilizing E. coli cells behind a dialysis membrane on a carbon paste electrode containing 2,3-dimethoxy-5-methyl-1,4-benzoquinone (Q(0)), produced a current for the electrocatalytic oxidation of glucose with Q(0) as an electron transfer mediator only after the addition of a trace amount of pyrroloquinoline quinone (PQQ), the cofactor of the enzyme. This allows a novel method of glucose measurements free from the interference of the redox active substances, if contained, in a sample solution. The glucose sensor was insensitive to dioxygen; the currents measured under anaerobic and aerobic conditions, and even under dioxygen saturated conditions were almost the same in magnitude at a given concentration of glucose over the range of 0.2-10 mM. Response time of the glucose sensor was 2 min to attain 90% level of the steady-state current. The E. coli modified glucose sensor was reusable when treated with ethylenediaminetetraacetic acid (EDTA). When E. coli cells were lyophilized, they could be stored at room temperature in a dry box for more than six months without loss of the catalytic activity.     

MEMBRANE LIFTING TECHNIQUE FOR RAPID DETECTION OF ESCHERICHIA COLI 0157:H7 IN INOCULATED GROUND MEATS

A membrane lifting technique was developed for direct rapid detection of Escherichia coli 0157:H7 in inoculated ground meats. Duplicate groups of 2 meatballs were inoculated with volumes of 0.1-ml of a serial dilution (1:10) of E. coli 0157:H7 or a mixed culture containing one strain of E. coli 0157:H7 and a non-0157:H7E. coli serotype (E. coli ATCC 25922). Each meatball was sampled by sandwiching with 2 pieces of nitrocellulose membranes and pressing against each other to the center of the meatball. The membranes were in contact with the meats for 10 min to lift the bacteria. The membranes were removed and incubated on MacConkey-sorbitol agar plates with the meat contact side up. After 18 h incubation at 37C, an immunostain was performed directly on the membranes for detection of the presence or absence of E. coli 0157:H7. This method was found to be sensitive enough to detect as few as 1 to 2 cells of E. coli 0157:H7 inoculated on surfaces of 18-g meatballs. This method might be used as a rapid presumptive test for E. coli 0157:H7.     

Common solvent toxicity: autoxidation of respiratory redox-cyclers enforced by membrane derangement

Unspecific biological effects of chemically diverse solvents strikingly reveal the unifying motif of oxidant toxicity both in higher organisms and in aerobic bacteria. In a few spectacular cases, solvent metabolites with oxidant properties were demonstrated, which however cannot explain extrahepatic toxicity, e.g. in muscle and nerve cells. A common source of solvent-inducible oxidants, by contrast, is suggested to be located in mitochondria or, more general, in membranes where the respiratory chain operates. Orderly respiration depends on membrane integrity, which is invariably compromised by exposure to most solvents and many other lipophils. In rat mitochondria, toluene-induced membrane derangement has been directly implicated with superoxide production, resulting from autoxidation of the membrane-located respiratory redox-cycler ubisemiquinone. A related mechanism may occur in bacteria: Exposure of Escherichia coli to lipophils such as ethanol, tetralin, indole, chlorpromazine and procaine, or to heat shock, induces anti-oxidant proteins, which are reliable indicators of increased oxidant levels. Although many molecular details remain to be elucidated, this review documents that oxidant toxicity of lipophilic compounds is a common physiological phenomenon correlated with derangement of membranes where respiratory processes take place. Subjective consequences of acute oxidant injury are probably the hangover from alcohol and nicotine consumption, and the sudden death from recreational solvent abuse. Suggestions concerning oxidants as major contributors to ageing remain unchallenged.     

Expression of the phospholipid-dependent Escherichia coli sn-1,2-diacylglycerol kinase in COS cells perturbs cellular lipid composition

The Escherichia coli sn-1,2-diacylglycerol (DAG) kinase has been successfully expressed in COS cells. The E. coli dgkA locus which contains the coding sequences for DAG kinase was subcloned into an eukaryotic expression vector, pMT2. COS cells transfected with the vector pMT2dgk expressed the DAG kinase as shown by Western analysis. Immunofluorescence studies revealed that the E. coli DAG kinase was prominently but not exclusively located in the endoplasmic reticulum. In addition, mixed micellar assays in beta-octyl glucoside revealed that membranes prepared from pMT2dgk-transfected COS cells contained over a 1500-fold increase in DAG kinase activity: 107 nmol/min/mg compared with only 0.067 nmol/min/mg for controls. DAG kinase activity from the E. coli enzyme was distinguished from endogenous COS cell activity based on differences in thermolability and the ability of the E. coli enzyme to use ceramide as a substrate. No ceramide kinase activity was detected in control COS cells, so the activity detected in pMT2dgk transfectants must have resulted from the expressed E. coli DAG kinase. The Km values for DAG kinase derived from E. coli and COS cells were nearly identical. Finally, transfected COS cells were labeled with [32P]Pi to investigate possible perturbations in lipid composition induced by the action of the E. coli DAG kinase. Ceramide (generated by the action of sphingomyelinase) was also used to clearly implicate the E. coli enzyme. Levels of ceramide phosphate increased more than 150-fold in pMT2dgk-transfected cells relative to controls. The results of these studies show that the E. coli enzyme expressed in COS cells is active and perturbs lipid composition in the intact cell system; the absolute lipid cofactor requirement of E. coli DAG kinase can be satisfied in COS cells.     

Effect of the Salmonella pathogenicity island 2 type III secretion system on Salmonella survival in activated chicken macrophage-like HD11 cells

In order to better identify the role of the Salmonella pathogenicity island 2 (SPI-2) type III secretion system (T3SS) in chickens, we used the well-known gentamicin protection assay with activated HD11 cells. HD11 cells are a macrophage-like chicken cell line that can be stimulated with phorbol 12-myristate 13-acetate (PMA) to exhibit more macrophage-like morphology and greater production of reactive oxygen species (ROS). Activated HD11 cells were infected with a wild-type Salmonella enterica subspecies enterica serovar Typhimurium (S. Typhimurium) strain, a SPI-2 mutant S. Typhimurium strain, a wild-type Salmonella enterica subspecies enterica serovar Enteritidis (S. Enteritidis) strain, a SPI-2 mutant S. Enteritidis strain, or a non-pathogenic Escherichia coli (E. coli) strain. SPI-2 mutant strains were found to survive as well as their parent strain at all time points post-uptake (PU) by the HD11 cells, up to 24 h PU, while the E. coli strain was no longer recoverable by 3 h PU. We can conclude from these observations that the SPI-2 T3SS of S. Typhimurium and S. Enteritidis is not important for survival of Salmonella in the activated macrophage-like HD11 cell line, and that Salmonella must employ other mechanisms for survival in this environment, as E. coli is effectively eliminated.     

Effect of chloramphenicol and cyanide on the increase in UV resistance of intracellular bacteriophage T1.

The effects of chloramphenicol and cyanide on the increase in UV resistance of intracellular phage T1 infecting cells of E. coli B or E. coli Bs-1 were investigated. The inhibitiors were added to the cells 3 min prior to infection and to the complexes of phage-bacteria 3.5 and 6.5 min after adsorption of phage by the cells. The data obtained are not in agreement with the suggestion that increase in UV resistance of intracellular phage is mainly due to the accumulation of phage DNA inside the host cells. It is suggested that a very important role in this resistance is played by the interaction of phage DNA with the cell membranes.     

The multiple functions of the thiol-based electron flow pathways of Escherichia coli: Eternal concepts revisited

Electron flow via thiols is a theme with many variations in all kingdoms of life. The favourable physichochemical properties of the redox active couple of two cysteines placed in the optimised environment of the thioredoxin fold allow for two electron transfers in between top biological reductants and ultimate oxidants. The reduction of ribonucleotide reductases by thioredoxin and thioredoxin reductase of Escherichia coli (E. coli) was one of the first pathways to be elucidated. Diverse functions such as protein folding in the periplasm, maturation of respiratory enzymes, detoxification of hydrogen peroxide and prevention of oxidative damage may be based on two electron transfers via thiols. A growing field is the relation of thiol reducing pathways and the interaction of E. coli with different organisms. This concept combined with the sequencing of the genomes of different bacteria may allow for the identification of fine differences in the systems employing thiols for electron flow between pathogens and their corresponding mammalian hosts. The emerging possibility is the development of novel antibiotics.     

Expression of SARS-coronavirus envelope protein in Escherichia coli cells alters membrane permeability

To promote viral entry, replication, release, and spread to neighboring cells, many cytolytic animal viruses encode proteins responsible for modification of host cell membrane permeability and for formation of ion channels in host cell membranes during their life cycles. In this study, we show that the envelope (E) protein of severe acute respiratory syndrome-associated coronavirus can induce membrane permeability changes when expressed in Escherichia coli. E protein expressed in bacterial and mammalian cells under reducing conditions existed as monomers, but formed homodimer and homotrimer under non-reducing conditions. Site-directed mutagenesis studies revealed that two cysteine residues of the E protein were essential for oligomerization, leading to induction of membrane permeability. This is the first report demonstrating that a coronavirus-encoded protein could modify membrane permeability in E. coli cells.     

Reconstitution of pyrroloquinoline quinone-dependent D-glucose oxidase respiratory chain of Escherichia coli with cytochrome o oxidase.

D-Glucose dehydrogenase is a pyrroloquinoline quinone-dependent primary dehydrogenase linked to the respiratory chain of a wide variety of bacteria. The enzyme exists in the membranes of Escherichia coli, mainly as an apoenzyme which can be activated by the addition of pyrroloquinoline quinone and magnesium. Thus, membrane vesicles of E. coli can oxidize D-glucose to gluconate and generate an electrochemical proton gradient in the presence of pyrroloquinoline quinone. The D-glucose oxidase-respiratory chain was reconstituted into proteoliposomes, which consisted of two proteins purified from E. coli membranes, D-glucose dehydrogenase and cytochrome o oxidase, and E. coli phospholipids containing ubiquinone 8. The electron transfer rate during D-glucose oxidation and the membrane potential generation in the reconstituted proteoliposomes were almost the same as those observed in the membrane vesicles when pyrroloquinoline quinone was added. The results demonstrate that the quinoprotein, D-glucose dehydrogenase, can reduce ubiquinone 8 directly within phospholipid bilayer and that the D-glucose oxidase system of E. coli has a relatively simple respiratory chain consisting of primary dehydrogenase, ubiquinone 8, and a terminal oxidase.     

Bacterial glutathione: a sacrificial defense against chlorine compounds.

Aerobic organisms possess a number of often overlapping and well-characterized defenses against common oxidants such as superoxide and hydrogen peroxide. However, much less is known of mechanisms of defense against halogens such as chlorine compounds. Although chlorine-based oxidants may oxidize a number of cellular components, sulfhydrl groups are particularly reactive. We have, therefore, assessed the importance of intracellular glutathione in protection of Escherichia coli cells against hydrogen peroxide, hypochlorous acid, and chloramines. Employing a glutathione-deficient E. coli strain (JTG10) and an otherwise isogenic glutathione-sufficient E. coli strain (AB1157), we find that glutathione-deficient organisms are approximately twice as sensitive to killing by both hydrogen peroxide and chlorine compounds. However, the mode of protection by glutathione in these two cases appears to differ: exogenous glutathione added to glutathione-deficient E. coli in amounts equal to those which would be present in a similar suspension of the wild-type bacteria fully restored resistance of glutathione-deficient bacteria to chlorine-based oxidants but did not change resistance to hydrogen peroxide. Furthermore, in protection against chlorine compounds, oxidized glutathione is almost as effective as reduced glutathione, implying that the tripeptide and/or oxidized thiol undergo further reactions with chlorine compounds. Indeed, in vitro, 1 mol of reduced glutathione will react with approximately 3.5 to 4.0 mol of hypochlorous acid. We conclude that glutathione defends E. coli cells against attack by chlorine compounds and hydrogen peroxide but, in the case of the halogen compounds, does so nonenzymatically and sacrificially.     

Flow-through immunofiltration assay system for rapid detection of E. coli O157:H7

A flow-through amperometric immunofiltration assay system based on disposable porous filter-membranes for rapid detection of Escherichia coli O157:H7 has been developed. The analytical system utilizes flow-through, immunofiltration and enzyme immunoassay techniques in conjunction with an amperometric sensor. The parameters affecting the immunoassay such as selection of appropriate filter membranes, membrane pore size, antibody binding capacity and the concentrations of immunoreagents were investigated and optimized. Non-specific adsorption of the enzyme conjugate was investigated and minimized. A sandwich scheme of immunoassay was employed and the immunofiltration system allows to specifically and directly detect E. coli cells with a lower detection limit of 100 cells/ml. The working range is from 100 to 600 cells/ml with an overall analysis time of 30 min. No pre-enrichment was needed. This immunosensor can be easily adapted for assay of other microorganisms and may be a basis for a new class of highly sensitive bioanalytical devices for rapid quantitative detection of bacteria.     

Evaluation of a microwire sensor functionalized to detect Escherichia coli bacterial cells

A functionalized microwire sensor based on dielectrophoresis (DEP) and antigen-antibody reaction was initially developed for sensitive and selective detection of E. coli O157:H7. The dynamics of gold-tungsten microwires were manipulated using an automated X-Y-Z stage and the sensing process included antibody immobilization and bacterial detection, and cell quantification. Antibodies were first immobilized on surface of the microwire to improve sensing specificity, and then coupled with DEP for capture of E. coli cells in a mixture of E. coli cells and non-conductive polystyrene beads. Afterward, fluorescein-conjugated secondary antibodies were applied to the wire for quantification of captured bacteria. Field Emission Scanning Electron Microscope (FESEM) figures and fluorescence intensities of bacteria on the wire validated the sensing mechanism. The entire immobilization and detection procedure could be completed within 30 min with simple operations. Performance of the microwire sensor was not significantly affected when conducted in orange juice. In addition, the detection limit of this sensor was about 5 bacterial cells per microwire in 1000 CFU/mL bacterial suspensions when the electric field generated at 3 MHz and 20 peak to peak voltage (V(pp)), and only targeted E. coli cells were concentrated and captured.