Variable specific activity of Escherichia coli beta-galactosidase in bacterial cells

Escherichia coli lacZ is a frequently employed reporter gene for the monitoring of gene expression and recombinant protein production due the simple determination of beta-galactosidase activity in both qualitative and quantitative assays. In the absence of either total or recombinant protein synthesis, we observed a lack of correlation between protein amount and enzymatic activity in both engineered and native beta-galactosidases in Escherichia coli cells. A delayed fading of beta-galactosidase activity compared with the rapid degradation of intact protein suggests a progressive increase in enzyme-specific activity during the life of the protein. This intriguing event does not involve solubilization from major protein aggregates and it occurs both in vivo and in cell extracts, but not in solutions of purified protein. Possible explanations for this activation are examined in the context of the assisted protein folding network and proteolytic degradation of misfolded proteins.     

Synthetic oligodeoxyribonucleotide probes to detect verocytotoxin-producing Escherichia coli in diseased pigs

Oligonucleotide probes constructed from the sequences published for Shiga-like toxin I (SLT-I) and Shiga-like toxin II (SLT-II) genes and antibody against the purified toxins were used to study the SLT (SLT-IIp) produced by porcine E. coli O138 and O139 strains. By DNA hybridization assays no homology was observed between SLT-I and SLT-IIp. By contrast the oligonucleotide probe derived from the slt-II A gene detected porcine strains of E. coli producing SLT-IIp and E. coli strains associated with human disease producing SLT-II. Homology of nucleotide sequences between SLT-IIp and SLT-II is reflected by serological cross-reactivity as demonstrated by a dot blot ELISA and neutralization of SLT-IIp with anti-SLT-II. The toxins were distinguishable in their ability to kill HeLa S-3 cells. The oligonucleotide probe and anti-SLT-II can facilitate identification of SLT-IIp producing E. coli to further clarify their role in diseased pigs.     

Evaluation of a novel chemical sensor system to detect clinical mastitis in bovine milk

Automatic detection of clinical mastitis is an essential part of high performance and robotic milking. Currently available technology (conductivity monitoring) is unable to achieve acceptable specificity or sensitivity of detection of clinical mastitis or other clinical diseases. Arrays of sensors with high cross-sensitivity have been successfully applied for recognition and quantitative analysis of other multicomponent liquids. An experiment was conducted to determine whether a multisensor system ("electronic tongue") based on an array of chemical sensors and suitable data processing could be used to discriminate between milk secretions from infected and healthy glands. Measurements were made with a multisensor system of milk samples from two different farms in two experiments. A total of 67 samples of milk from both mastitic and healthy glands were in two sets. It was demonstrated that the multisensor system could distinguish between control and clinically mastitic milk samples (p=0.05). The sensitivity and specificity of the sensor system (93 and 96% correspondingly) showed an improvement over conductivity (56 and 82% correspondingly). The multisensor system offers a novel method of improving mastitis detection.     

Monitoring growth of Escherichia coli using a two-channel optical sensor

Summary The growth of Escherichia coli strain TG 1 was monitored, measuring simultaneously the culture fluorescence and the 360° reflection at 578 nm with a two-channel optical sensor. It was observed that the culture fluorescence at 366 nm excitation was approximately three times higher than the NADH fluorescence of washed E. coli cells whereas the 360° reflection at 578 nm was comparable. The reason for this effect was found to be the accumulation of riboflavin in the cultivation liquid of the E. coli cells being equal to approximately 0.05 mg/g biomass. In shaken batch cultivations of the same strain the amount of riboflavin in the cell-free cultivation liquid correlated with the biomass being a very sensitive indicator of E. coli growth.Correspondence to: W. S. Kunz     

产超广谱β-内酰胺酶大肠埃希菌对喹诺酮类抗菌药物的耐药性检测

目的 了解临床分离产超广谱β-内酰胺酶（ESBLs）大肠埃希菌对喹诺酮类等抗菌药物的耐药性。方法 NCCLS表型确证试验（纸片增强法）检测出临床分离大肠埃希菌中产ESBLs菌株,琼脂稀释法测定产ESBLs菌株对喹诺酮类等抗菌药物的耐药性。结果 临床分离大肠埃希菌中产ESBLs菌株的检出率为40．2％（92／229）,产ESBLs菌株以尿标本多见,对6种喹诺酮类抗菌药物的耐药率均在89％以上,哌拉西林的耐药率为100％;对头孢噻肟、头孢他啶和哌拉西林-三唑巴坦的耐药率分别为77．2％、1．1％和21．7％,对亚胺培南极其敏感,耐药率为0％。结论 产ESBLs大肠埃希菌发生率较高,对喹诺酮类抗菌药物耐药显著,临床应加强检测和监测。     

Application of a flow-type antibody sensor to the detection of Escherichia coli in various foods

A flow-type biosensor system which uses a broad-spectrum anti-Escherichia coli antibody and quartz crystal microbalance as biological component and transducer was developed. Biosensor responses were initiated by injecting viable E. coli suspensions through a flow cell and the sensor system was optimized for response time according to flow rate and injection time, followed by the measurement of responses for various E. coli strains. As expected, the sensor system showed a characteristic broad binding feature against E. coli strains. A linear sensor response in double-logarithmic scale was observed for the microbial suspensions ranging from 1.7 x 10(5) to 8.7 x 10(7) CFU/ml. Sample measurements could be done within 20-30 min after Stomacher treatment followed by spiking or enrichment.     

Adhesion of enteropathogenic Escherichia coli to host cells

Enteropathogenic Escherichia coli (EPEC) adhere to the intestinal mucosa and to tissue culture cells in a distinctive fashion, destroying microvilli, altering the cytoskeleton and attaching intimately to the host cell membrane in a manner termed the attaching and effacing effect. Typical EPEC strains also form three-dimensional microcolonies in a pattern termed localized adherence. Attaching and effacing, and in particular intimate attachment requires an outer membrane adhesin called intimin, which binds to the translocated intimin receptor, Tir. Tir is produced by the bacteria and delivered to the host cell via a type III secretion system. In addition to this well-established adhesin-receptor pair, numerous other adhesin interactions between EPEC and host cells have been described including those between intimin and cellular receptors and those involving a bundle-forming pilus and flagella and unknown receptors. Much additional work is needed before a full understanding of EPEC adhesion to host cells comes to light.     

Branched Escherichia coli cells

We report that the normally rod-shaped bacterium Escherichia coli can form branched cells. These were found in strains in which chromosome replication or nucleoid segregation was disturbed, e.g. in minB mutants, intR1 strains, and in strains exhibiting stable DNA replication. Often, chromosome DNA was found to be located in the branch point of the cells. The branching frequency was dependent upon the growth medium: in rich medium no branched cells were found, whereas in minimal medium containing acetate and casamino acids the frequency of branched cells was increased. The genetic background of the strains also affected the tendency to branch. Furthermore, electron microscopy of thin-sectioned branched cells revealed additional membrane-like structures, which were not observed in wild-type cells. Finally, the branched cells are compared with bacteria that normally branch, and probable causes for branching in E. coli are discussed.     

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.     

Structure of an Escherichia coli bacterial suspension]

The structure of bacterial suspensions of Escherichia coli M-17 at the counting concentrations of the cells 10(7), 10(8), 10(9) i/ml and in the temperature range of (18-50) degrees C has been investigated by means of orientational conductometric, electron microscopic and UV-spectroscopic methods. On the basis of experimental relationships of the anisotropy of suspensions electric conductivity upon the intensity of a sinusoidal electric field and relaxation of anisotropy after switching off the field the function of the distribution of bacteria with respect to their sizes was evaluated at different temperatures and concentrations. The conductometric function of bacteria distribution is in a good agreement with the analogous function obtained with the help of the electron microscope. In accordance with the functions the suspension of E. coli contained three kinds of cells: high electronic density, low electronic density bacteria and bacteria aggregates. Relative amounts of every kind of bacteria depended on temperature and concentration of cells. The minimum of bacteria aggregates and maximum of low electronic density cells were obtained in the temperature range of (32-42) degrees C. This fact could be explained by the activation of the transport membrane systems in this temperature range. This hypothesis was confirmed by the UV-spectroscopic method.     

Microbial sensor for penicillins using a recombinant strain of Escherichia coli.

E. coli harboring the multicopy plasmid pBR327, which codes for beta-lactamase synthesis, was immobilized on acetylcellulose membranes. These were placed in the vicinity of a flat pH electrode, thus constituting a penicillin biosensor based on the detection of changes in pH using a reference pH electrode. A response time of 8 min was obtained with at least 2 mg of cells cm-2 of membrane. A linear detection range between 5 and 30 mM of penicillin was achieved. Buffering capacity decreased the sensitivity, but to a lower extent as when compared with probes using purified enzyme. The sensor was completely stable for at least 13 days and proved to be useful for penicillin estimation in complex media such as fermentation broths and milk.     

Globin coupled sensor and photosensitive signaling network--receptors of monochromatic light in bacteria Escherichia coli cells

The effect of He-Ne laser radiation in wide range of intensities on E. coli cells division was studied in dependence on dose of light, on time of irradiation as well as on cAMP content in initial bacterial culture. Two maxima in E.coli growth stimulation vs dose or irradiation time curves were observed: first--irradiation time-independent, near 55 J/m2, second--dose-independent, near 100 s. It was proposed that latter maximum reflects kinetic parameters of photosensitive signaling network, and the former one reflects redox state of globin-coupled direct photosensor--EcDOS phosphodiesterase cAMP.     

A colony blot immunoassay to detect enteroinvasive Escherichia coli and Shigella in water samples

AIMS: The aim of the study was to develop a colony blot immunoassay to detect Shigella and enteroinvasive Escherichia coli (EIEC) in water. METHODS AND RESULTS: Spiked samples were filtered through nitrocellulose membranes. Colony prints on the filters were tested with a monoclonal antibody specific to IpaC, an antigen coded by the invasion plasmid of Shigella and EIEC. Invasive pathogens could be successfully detected with the technique, even in the presence of a large number of non-pathogenic bacterial cells. The method was significantly more sensitive in identifying pathogen-containing samples then the traditional culture-based approach. CONCLUSION: The IpaC-specific colony blot immunoassay is an inexpensive method for identifying the aetiological agents of bacillary dysentery in water samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The technique could be particularly useful in detecting enteroinvasive E. coli which often remains undetected by bio- and serotyping.     

A biotinylated DNA probe to detect bacterial cells in artificially contaminated foodstuffs

A biotin-labelled DNA probe was used in a dot-blot hybridization test to demonstrate the presence of Escherichia coli in a variety of artificially contaminated foodstuffs. Positive hybridization was detected by using a streptavidine/polyalkaline phosphatase conjugate to generate an insoluble coloured precipitate in the presence of an appropriate dye. The colour intensity was measured with a computer-controlled image analysis system which assessed objectively the hybridization signal produced by each sample. The method was capable of distinguishing positive hybridization at cell concentrations exceeding 10(4) cells/dot-blot, equivalent to 2 x 10(7) cells/g food, and had none of the drawbacks normally associated with the use of radioactively labelled DNA in hybridization techniques. The procedure is highly specific and takes less than 30 h. Many samples can be screened simultaneously and the procedure can be used to detect any species for which a suitable DNA probe is available.     

A functional assay in Escherichia coli to detect non-assisted interaction between galactose repressor dimers

Among the Escherichia coli operons repressed from multiple sites on DNA, the galactose operon is unique: its repression requires an auxiliary protein, HU, to assist cooperative repressor binding to two distant DNA sites. Here we show that GalR can still mediate repression from distant sites in an artificial and simplified regulatory region which totally disturbs the organisation of the natural interactions. This simple and unexpected cooperation of a protein incapable of self-association in solution might be involved in regulation of the gal operon. Furthermore, the assay may be generalised to detection of rather weak cooperative interactions between DNA-bound proteins.     

Evaluation of Enzyme-Labeled Oligonucleotide Probes to Identify Enterohaemorrhagic Escherichia coli

Alkaline phosphatase-conjugated oligonucleotide probes were developed to detect the gene coding for Vero toxin 1 (VT1) and Vero toxin 2 (VT2). Using these probes, 3 hr was enough to detect VT genes when suspicious colonies of enterohaemorrhagic Escherichia coli (EHEC) were obtained on an agar plate. The results of a hybridization test with 144 isolates of EHEC O157 and one isolate of Shigella dysenteriae Type 1 agreed exactly with the immunological detection, reversed passive latex agglutination (RPLA) test, of VTs in their culture supernatants. The sensitivity levels of these probes for the detection of VT genes were 100%. The specificity of these probes were also tested with a total of 1,002 strains of Escherichia coli other than EHEC and 8 strains of Shigella sp. other than Shigella dysenteriae Type 1; the results showed 100% specificity.     

Identification of a ribosomal ATPase in Escherichia coli cells

Eukaryotic ribosomes harbor an ATPase activity that has been shown to be essential for translation elongation in some lower fungi. Here we report the first identification of a ribosome bound ATPase, RbbA, in E. coli cells. RbbA accounts for most of the ATPase activity associated with 70S ribosomes and 30S ribosomal subunits. Both native and recombinant RbbA were purified and shown to possess ribosome-dependent ATPase activities and to stimulate polyphenylalanine synthesis in vitro. Biochemically, RbbA is similar to the fungi-specific translation elongation factor 3 (EF-3) and cross-reacts with antibody raised against EF-3. The gene encoding RbbA is identified as ORF yhih and the predicted RbbA amino acid sequence is 40% similar to that of the C-terminal half of EF-3. The discovery of a ribosomal ATPase in a prokaryotic cell suggests a common, conserved function for these proteins in translation.