Detection of immunomagnetically captured Escherichia coli O157:H7 by antibody-conjugated alkaline phosphatase.

A rapid and sensitive detection process for Escherichia coli O157:H7 was developed using alkaline phosphatase (APase)-labeled anti-E. coli O157 antibodies to tag the targeted bacteria. Immunomagnetic beads or antibody-labeled streptavidin-coated magnetic beads were then used to capture the APase-tagged E. coli. Immunomagnetically captured bacteria were washed and distributed into microplates or optical cuvettes. The enzyme-catalyzed hydrolysis of p-nitro-phenol phosphate in alkaline solutions was then followed. Less than 1000 cfu/ml of E. coli O157:H7 could be detected. This approach was applied to detect the bacteria artificially spiked in beef hamburgers. Less than 1 cfu/g of E. coli O157:H7 produced a significant response after cultural enrichment for 4-6 h at 37 degrees C.

Immunomagnetic separation methods for the isolation of Campylobacter jejuni from ground poultry meats.

Campylobacter jejuni is now recognized as a leading foodborne pathogen, for which poultry products constitute the main transmission route. Two alternative immunomagnetic beads (IMB) were tested for direct detection of C. jejuni ATCC 35918 in artificially inoculated ground poultry meats and culture suspension. Polyclonal anti-Campylobacter antibodies were used to coat tosylactivated Dynabeads. The same antibodies conjugated with biotin were used to label streptavidin-coated beads. After these beads were incubated with inoculated poultry slurry or culture suspension, Campylobacter-bead complexes were separated from other components with a magnet. The capture efficiency was tested by plating bead-captured cells and unbound cells in the supernatant onto Karmali agar. The effects of different coating procedures, incubation time (60, 90, 120 min), numbers of immunomagnetic beads (10(6) to 10(7)/ml) and innoculum levels (10(3) to 10(7) CFU/g or ml) were determined. Without pre-enrichment, this approach could detect 10(4) CFU/g of ground poultry meats. These methods represent a new approach to extracting, concentrating and isolating Campylobacter spp. directly from foods.

Effects of chilling on sampling of bacteria attached to swine carcasses.

Two microbiological sampling techniques, excision and sponge swabbing, were compared by determining counts of aerobic bacteria, coliforms and injured coliforms from 20 de-haired swine carcasses before and after chilling. Excised jowl skin produced significantly greater counts of the three types of bacteria than sponge swabs. Aerobic bacteria, coliforms and injured coliforms recovered by sponge swabbing carcasses before chilling were 11.6%, 0.9% and 11.0% of excised samples, respectively; the corresponding percentages recovered after chilling were 23.9%, 11.1% and 5.0%. Numbers of all bacteria present on the post-chill carcasses were substantially lower than on the pre-chill carcasses. Excision usually produced more countable plates for coliforms and injured coliforms on chilled carcasses than sponge swabbing and therefore, is more suitable in estimating low numbers of faecal bacteria on chilled carcasses. To explore the possible structural bases for these findings, skin samples were inoculated with 10(2)-10(7) cfu cm(-2) faecal bacteria and examined by scanning electron microscopy. Chilled samples showed bacteria and biofilm embedded in superficial crevices, which underlies a possible reason for the lower recovery of bacterial cells by the sponge swabbing. The study indicates that the differences between sampling techniques may be a result of the chilling process of swine carcasses.

Minimum detectable level of Salmonellae using a binomial-based bacterial ice nucleation detection assay (BIND).

A modified bacterial ice nucleation detection (BIND) assay was used for rapid and sensitive detection of several Salmonella species. For the BIND assay, Salmonella cells are infected with bacteriophage genetically modified to contain DNA encoding an ice nucleation protein (INP). After infection, de novo protein synthesis occurs and INPs are incorporated into the outer membrane of the organism. After supercooling (-9.3 degrees C), only buffer solutions containing transfected salmonellae freeze, causing a phase-sensitive dye to change color. This technique, and a probability-based protocol modification, provided quantitative detection with a minimum detectable level (MDL) of 2.0 +/- 0.3 S. enteritidis cells/mL in buffer (about 3 h). The MDLs for S. typhimurium DT104 and S. abaetetuba were 4.2 +/- 0.2 and 11.1 +/- 0.4 cells/mL, respectively. Using salmonellae-specific immunomagnetic bead separation technology in conjunction with the modified BIND protocol, we achieved an MDL of about 4.5 S. enteritidis cells/mL with an apparent capture efficiency of 56%.

Immobilization of horseradish peroxidase in cross-linked phyllosilicates: conditions and characterizations.

An innovative immobilization procedure was developed for intercalation of enzymes into dispersed phyllosilicates which were cross-linked with silicates resulting from the hydrolysis of tetramethyl orthosilicate. Donor:hydrogen-peroxide oxidoreductase intercalative immobilized in the cross-linked phyllosilicate exhibited a similar or higher activity than the free enzyme. The kinetic properties of peroxidase were unaffected by intercalative immobilization. Different factors, including drying methods, particle size, surface cations of the phyllosilicate and ratio of phyllosilicate to tetramethyl orthosilicate, were investigated to optimize immobilization conditions. The immobilized peroxidase exhibited similar kinetic properties to the free enzyme and good storage stability.

Use of a light-addressable potentiometric sensor for the detection of Escherichia coli O157:H7.

We describe the development of an immunoligand assay (ILA) in conjunction with a light-addressable potentiometric sensor (LAPS) for the rapid detection of Escherichia coli O157:H7 cells in buffered saline. The ILA protocol consists of "sandwiching" bacterial analyte between biotinylated and fluoresceinated antibodies, indirect enzyme labeling of the bacteria with urease-labeled anti-fluorescein antibody, and active capture of the immune complex at a biotinylated bovine serum albumin-blocked nitrocellulose filter membrane with streptavidin. Using live E. coli O157:H7, the efficiency of the ILA was compared using various ratios of the biotinylated and fluoresceinated antibodies. Simultaneous addition of equimolar biotinylated and fluoresceinated antibodies effected optimal urease labeling and subsequent active capture of the bacteria in the ILA. Equimolar concentrations of the antibodies were varied to achieve optimal LAPS detection response for the live bacteria. Using ILA with LAPS, a minimum detectable level of ca. 7.1 x 10(2) cells/ml of heat-killed or ca. 2.5 x 10(4) cells/ml of live E. coli O157:H7 bacteria was achieved in Tris-buffered saline in an assay time of ca. 45 or ca. 30 min, respectively.

Effectiveness of cross-linked phyllosilicates for intercalative immobilization of soybean lipoxygenase.

A novel procedure was developed to intercalate enzymes into dispersed phyllosilicates that were cross-linked with silicate polymers formed by the hydrolysis of tetramethyl orthosilicate (TMOS). Lipoxygenase (LOX) intercalated into cross-linked phyllosilicates exhibited high enzymatic activity. The enzyme-phyllosilicate composite prepared by this procedure had an improved pore network. Alkylamines were used to occupy the charge sites of the phyllosilicate, which increased the hydrophobicity of the phyllosilicate and reduced charge-charge interaction between LOX and the phyllosilicate. The amount of macropores and the enzymatic activity of the lipoxygenase-phyllosilicate composites increased with an increase in the ratio of trimethylammonium (TMA)-phyllosilicate to cross-linking reagent TMOS. LOX intercalatively immobilized into phyllosilicates displayed good storage stability and reusability at ambient temperature.

Effects of Bafilomycin A1 and Metabolic Inhibitors on the Maintenance of Vacuolar Acidity in Maize Root Hair Cells.

Proton pumps of tonoplast membranes have been studied extensively in vitro, but data concerning their regulation in vivo are lacking. Effects of either anoxia, or the addition of KCN, 2-deoxy-d-glucose (deoxy-glucose), or bafilomycin-A1 (BAF) on vacuolar pH of maize (Zea mays L.) root hair cells were followed by fluorescence microscopy after loading of 2[prime]7[prime]-bis-(2-carboxyethyl)-5-(and-6) carboxyfluorescein. Root hair cells were able to maintain vacuolar acidity for at least 2 h in the presence of either 10 mM KCN or 50 mM deoxy-glucose or during anoxia. Treatments with either deoxy-glucose or KCN reduced total tissue ATP more than anoxia. ADP accumulated during anoxia and treatment with KCN as detected by in vivo 31P-NMR spectroscopy, but not during deoxy-glucose treatment. With control roots and roots treated with deoxy-glucose, the presence of BAF, a specific inhibitor of the V-type ATPase, caused alkalization of the vacuolar pH. However, either in the presence of KCN or under anoxic conditions, BAF was relatively ineffective in dissipating vacuolar acidity. Therefore, under anoxia or in the presence of KCN, unlike the situation with air or deoxy-glucose, the V-type ATPase apparently is not required for maintenance of vacuolar acidity.

Effects of solubilization on the inhibition of the p-type ATPase from maize roots by N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline.

The biochemical events utilized by transport proteins to convert the chemical energy from the hydrolysis of ATP into an electro-chemical gradient are poorly understood. The inhibition of the plasma membrane ATPase from corn (Zea mays L.) roots by N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) was compared to that of ATPase solubilized with N-tetradecyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate (3-14) to provide insight into the minimal functional unit. The chromatographic behavior of the 3-14-solubilized ATPase activity during size exclusion chromatography and glycerol gradient centrifugation indicated that the solubilized enzyme was in a monomeric form. Both plasma membrane-bound and solubilized ATPase were inhibited by EEDQ in a time- and concentration-dependent manner consistent with a first-order reaction. When the log of the reciprocal of the half-time for inhibition was plotted as a function of the log of the EEDQ concentration, straight lines were obtained with slopes of approximately 0.5 and 1.0 for membrane-bound and 3-14-solubilized ATPase, respectively, indicating a change in the number of polypeptides per functional ATPase complex induced by solubilization with 3-14.

Effects of temperature on the coupled activities of the vanadate-sensitive proton pump from maize root microsomes.

The mechanism by which proton transport is coupled to ATP hydrolysis by vanadate-sensitive pumps is poorly understood. The effects of temperature on the activities of the vanadate-sensitive ATPase from maize (Zea mays) roots were assessed to provide insight into the coupling mechanism. The initial rate of proton transport had a bell-shaped dependence on temperature with an optimal range between 20 and 30 degrees C. However, the rate of vanadate-sensitive ATP hydrolysis increased as the temperature was raised from 4 to 43 degrees C. The differential sensitivity of proton transport to temperatures above 30 degrees C was also observed when the ATPase was reconstituted into dioleoylphosphatidylcholine vesicles. Inhibition of proton transport with temperatures above 30 degrees C was associated with higher rates of proton leakage from the membranes. In addition, proton transport was more inhibited than ATP hydrolysis at temperatures below 10 degrees C. Reduced rates of proton transport at lower temperatures were not associated with higher rate of proton conductivity across the membranes. Therefore, the preferential inhibition of proton transport at temperatures below 10 degrees C may reflect an effect of temperature on the coupling between proton transport and ATP hydrolysis within the vanadate-sensitive ATPase.

N-Cyclo-N'-(4-Dimethylamino-alpha-Naphthyl)Carbodiimide Inhibits Membrane-Bound and Partially Purified Tonoplast ATPase from Maize Roots.

Certain carboxylic acid groups within the primary structure of proton translocating proteins are thought to be involved in the proton pathway. In this report, the effects of a lipophilic carboxylic acid reactive reagent, N-cyclo-N'(4-dimethylamino-alpha-naphthyl)carbodiimide (NCD-4), on the two types of proton pumps in maize (Zea mays L.) root microsomes were investigated. NCD-4 was found to inhibit the vacuolar-type H(+)-ATPase in microsomal preparations; however, the plasma membrane-type H(+)-ATPase was unaffected. The H(+)-ATPase in highly purified tonoplast vesicles was also inhibited by NCD-4. Inhibition was dependent on the concentration and length of exposure to the reagent. However, there was little, if any, increase in the fluorescence of treated vesicles, indicating few carboxylic acid residues were reacting. Inhibition of the tonoplast H(+)-ATPase by NCD-4 was examined further with a partially purified preparation. The partially purified H(+)-ATPase also showed sensitivity to the NCD-4, supporting the hypothesis that this carboxylic acid reagent is an inhibitor of the tonoplast ATPase from maize roots.

In vivo 133Cs-NMR a probe for studying subcellular compartmentation and ion uptake in maize root tissue.

Three 133Cs-NMR signals were observed in the spectra of CsCl-perfused and CsCl-grown maize seedling root tips. Two relatively broad lower field resonances were assigned to the subcellular, compartmented Cs+ in the cytoplasm and vacuole, respectively. The rate of area increase of the broader cytoplasmic Cs resonance was about 9-times faster than that of the vacuolar signal during the first 300 min of tissue perfusion with CsCl. In addition, the spin lattice relaxation time of the cytoplasmic Cs resonance was approx. 3-times shorter than that of the extracellular resonance, while the Cs+ signal associated with the metabolically less active vacuolar compartment exhibited a relaxation time comparable to that of the extracellular signal. 133Cs spectra of excised, maize root tips and excised top sections of the root adjacent to the kernel, each grown in 10 mM CsCl showed a difference in the relative areas of the Cs resonance corresponding to the distinct cytoplasm/vacuole volume ratio of these well differentiated sections of the root. The high correlation of counterion concentration with 133Cs chemical shifts suggested that the larger downfield shift exhibited by the cytoplasmic confined Cs+ was due principally to the higher ionic strength and protein content in this compartment. Such observations indicate that 133Cs-NMR might be employed for studying ionic strength, and osmotic pressure associated chemical shifts and the transport properties of Cs+ (perhaps as an analogue for K+) in subcellular compartments of plant tissues.

Differential Inhibition of Tonoplast H-ATPase Activities by Fluorescamine and Its Derivatives.

Corn (Zea mays L.) root tonoplast vesicles were treated with the primary-amine specific reagent, fluorescamine (FL). Modification by FL caused a differential inhibition to the coupled activities of tonoplast H(+)-ATPase. Within the range of 0 to 5 micromoles of FL per milligram of protein, the proton pumping rate was significantly reduced but ATP hydrolysis was only slightly affected. Yet, the membrane H(+) leakage during the pumping stage increased only slightly. FL treatment resulted in (a) a decrease in amine containing phospholipids and (b) an insertion of multiple H-bonding moieties into the membrane. To test which of these two possible effects were responsible for inhibition, FL derivatives of benzylamine, butylamine, and phenylalanine were synthesized. It was found that the acyclic derivatives with high H-bonding potential at concentrations of 10 micromolar inhibited proton pumping by 50% without a significant effect on ATP hydrolysis. Cyclic derivatives were largely ineffectual. Proton leakage during pumping was not affected by these acyclic modifiers. Membrane fluidity, as measured by the polarization of diphenyl hexatriene, decreased upon treatment with either FL or its derivatives. The results suggest that the proton pumping is indirectly linked to ATP hydrolysis in the tonoplast vesicles, and the link between these processes is apparently weakened by the presence of acyclic fluorescamine derivatives in the membrane.

Phosphate Uptake by Excised Maize Root Tips Studied by in VivoP Nuclear Magnetic Resonance Spectroscopy.

The extent of phosphate uptake measured by the relative changes in cytoplasmic Pi, vacuolar Pi, ATP, glucose-6-phosphate, and UDPG was determined using in vivo(31)P nuclear magnetic resonance spectroscopy. Maize (Zea mays) root tips were perfused with a solution containing 0.5 or 1.0 millimolar phosphate at pH approximately 6.5 under different conditions. In the aerated state, phosphate uptake resulted in a significant increase (>80%) in vacuolar Pi, but cytoplasmic Pi only transiently increased by 10%. Under N(2), the cytoplasmic Pi increased approximately 150% which could be attributed to a large extent to the breakdown of ATP, sugar phosphates and UDPG. Vacuolar Pi increased but only to the extent of approximately 10% of that seen under aerobic conditions. 2-deoxyglucose pretreatment was utilized to decrease the level of cytoplasmic Pi. When pretreated with the 2-deoxyglucose, the excised maize roots absorbed phosphate from the perfusate with a significant increase in the cytoplasmic Pi. The increase could only be traced to external phosphate since the concentrations of other phosphorus containing species remained constant during the uptake period. With 2-deoxyglucose pretreatment, phosphate uptake under anaerobic conditions was substantially inhibited with only the vacuolar phosphate showing a slight increase. When roots were treated with carbonyl cyanide m-chlorophenyl hydrazone, no detectable Pi uptake was found. These results were used to propose a H(+)-ATPase related transport mechanism for phosphate uptake and compartmentation in corn root cells.

Inhibition of cell wall-associated enzymes in vitro and in vivo with sugar analogs.

Sugar analogs were used to study the inhibition of cell wall-associated glycosidases in vitro and in vivo. For in vitro characterization, cell walls were highly purified from corn (Zea mays L.) root cortical cells and methods were developed to assay enzyme activity in situ. Inhibitor dependence curves, mode of inhibition, and specificity were determined for three sugar analogs. At low concentrations of castanospermine (CAS), 2-acetamido-1,5-imino-1,2,5-trideoxy-d-glucitol, and swainsonine, these inhibitors showed competitive inhibition kinetics with beta-glucosidase, beta-GIcNAcase, and alpha-mannosidase, respectively. Swainsonine specifically inhibited alpha-mannosidase activity, and 2-acetamido-1,5-imino-1,2,5-trideoxy-d-glucitol specifically inhibited beta-N-acetyl-hexosamindase activity. However, CAS inhibited a broad spectrum of cell wall-associated enzymes. When the sugar analogs were applied to 2 day old corn seedlings, only CAS caused considerable changes in root growth and development. To ensure that the concentration of inhibitors used in vitro also inhibited enzyme activity in vivo, an in vivo method for measuring cell wall-associated activity was devised.

Characterization of the Effects of Divalent Cations on the Coupled Activities of the H-ATPase in Tonoplast Vesicles.

The substrate requirement of the H(+)-ATPase in purified corn root tonoplast vesicles was investigated. The coupled activities, ATP hydrolysis and proton pumping, were simultaneously supported only by Mg(2+) or Mn(2+). The presence of Ca(2+) or Ba(2+) did not significantly affect the coupled activities. The addition of Cd(2+), Co(2+), Cu(2+), and Zn(2+) inhibited both the hydrolysis of Mg-ATP and the proton transport. However, the inhibition of proton pumping was more pronounced. Based on equilibrium analysis, both ATP-complexed and free forms of these cations were inhibitory. Inhibition of the hydrolysis of Mg-ATP could be correlated to the concentrations of the ATP-complex of Zn. On the other hand, the free Cu(2+) and Co(2+) were effective in inhibiting hydrolysis. For proton pumping, the ATP complexes of Co(2+), Cu(2+), and Zn(2+) were effective inhibitors. However, this inhibition could be further modulated by free Co(2+), Cu(2+), and Zn(2+). While the equilibrium concentrations of Cd-ATP and free Cd(2+) were not estimated, the total concentration of this cation needed to inhibit the coupled activities of the H(+)-ATPase was found to be in the range of 10 to 100 micromolars. The presence of free divalent cations also affected the structure of the lipid phase in tonoplast membrane as demonstrated by the changes of emission intensity and polarization of incorporated 1,6-diphenyl-1,3,5-hexatriene. The differential inhibition caused by these cations could be interpreted by interactions with the protogenic domain of the membrane as previously proposed in "indirect-link" mechanism.

In VivoP NMR Spectroscopic Studies of Soybean Bradyrhizobium Symbiosis: I. Optimization of Parameters.

(31)P NMR spectroscopy was used to study in vivo the symbiotic state established between soybean (Glycine max [L.] Merr. cv Williams) and Bradyrhizobium japonicum (USDA 110 and 138). Different experimental conditions were used to maintain perfused, respiring detached or attached nodules in an NMR magnet. The pH of the perfusion medium affected the cytoplasmic pH and the resolution of the spectra. The internal Pi content and distribution were assessed as a function of nodule age and green-house growth conditions and the rate of glucose and 2-deoxyglucose uptake into nodules in split and intact states. The major metabolites (glucose-6-P, fructose-1,6-diP, P-choline, Pi, NTP, UDP-glc, and NAD) were readily identified from (31)P NMR spectra of perchloric acid extracts of nodules with the exception of one unknown phosphorus metabolite. Nodules stressed by glucose deprivation demonstrated movement of Pi between the vacuole and cytoplasmic compartments not previously observed in (31)P NMR studies.

Phospholipid requirement of the vanadate-sensitive ATPase from maize roots evaluated by two methods.

The activation of the vanadate-sensitive ATPase from maize (Zea mays L.) root microsomes by phospholipids was assessed by two different methods. First, the vanadate-sensitive ATPase was partially purified and substantially delipidated by treating microsomes with 0.6% deoxycholate (DOC) at a protein concentration of 1 milligram per milliliter. Vanadate-sensitive ATP hydrolysis by the DOC-extracted microsomes was stimulated up to 100% by the addition of asolectin. Of the individual phospholipids tested, phosphatidylserine and phosphatidylglycerol stimulated activity as much as asolectin, whereas phosphatidylcholine did not. Second, phospholipid dependence of the ATPase was also assessed by reconstituting the enzyme into proteoliposomes of differing phospholipid composition. In these experiments, the rate of proton transport and ATP hydrolysis was only slightly affected by phospholipid composition. DOC-extracted microsomes reconstituted with dioleoylphosphatidylcholine had rates of proton transport similar to those found with microsomes reconstituted with asolectin. The difference between the two types of assays is discussed in terms of factors contributing to the interaction between proteins and lipids.

Temperature dependence and mercury inhibition of tonoplast-type H+-ATPase.

The effects of changing temperature on ATP hydrolysis and proton pumping associated with the H+-ATPase of tonoplast membrane vesicles isolated from the maize root microsomal fraction were determined. In the range 5 to 45 degrees C, the maximal initial rate of ATP hydrolysis obeyed a simple Arrhenius model and the activation energy determined was approximately 14 kcal/mol. On the other hand, the initial proton pumping rate showed a bell-shaped temperature dependence, with maximum activity around 25 degrees C. Lineweaver-Burke analysis of the activities showed that the Km of ATP hydrolysis, unlike that of proton pumping, was relatively insensitive to temperature changes. Detailed kinetic analysis of the proton pumping process showed that the increase in membrane leakage to protons during the pumping stage constituted a major reason for the decreased transport. Nitrate-sensitive ATPase activities of the tonoplast vesicles were found to be inhibited by the presence of micromolar concentrations of Hg2+. The proton pumping process was more sensitive to the presence of Hg2+. Double-reciprocal analysis of kinetic data indicated that Hg2+ was a noncompetitive inhibitor of proton pumping but was an uncompetitive inhibitor of ATP hydrolysis. Further kinetic analysis of Hg2+ effects revealed that the lower proton transport did not result from enhanced membrane leakage but rather from reduced coupling between H+ pumping and ATP hydrolysis.

Effects of multivalent cations on cell wall-associated Acid phosphatase activity.

Primary cell walls, free from cytoplasmic contamination were prepared from corn (Zea mays L.) roots and potato (Solanum tuberosum) tubers. After EDTA treatment, the bound acid phosphatase activities were measured in the presence of various multivalent cations. Under the conditions of minimized Donnan effect and at pH 4.2, the bound enzyme activity of potato tuber cell walls (PCW) was stimulated by Cu(2+), Mg(2+), Zn(2+), and Mn(2+); unaffected by Ba(2+), Cd(2+), and Pb(2+); and inhibited by Al(3+). The bound acid phosphatase of PCW was stimulated by a low concentration but inhibited by a higher concentration of Hg(2+). On the other hand, in the case of corn root cell walls (CCW), only inhibition of the bound acid phosphatase by Al(3+) and Hg(2+) was observed. Kinetic analyses revealed that PCW acid phosphatase exhibited a negative cooperativity under all employed experimental conditions except in the presence of Mg(2+). In contrast, CCW acid phosphatase showed no cooperative behavior. The presence of Ca(2+) significantly reduced the effects of Hg(2+) or Al(3+), but not Mg(2+), to the bound cell wall acid phosphatases. The salt solubilized (free) acid phosphatases from both PCW and CCW were not affected by the presence of tested cations except for Hg(2+) or Al(3+) which caused a Ca(2+)-insensitive inhibition of the enzymes. The induced stimulation or inhibition of bound acid phosphatases was quantitatively related to cation binding in the cell wall structure.