Enzyme-Linked Immunosorbent Assays for Detection of Xylem-Limited Bacteria: Use of Trinder Reagent

Quantitation and detection of xylem-limited bacteria with an enzyme-linked immunosorbent assay with a peroxidase conjugate is described. The use of the Trinder reagent (4-aminoantipyrine) allows the determination of extremely small quantities of peroxidase with no precipitate formation or inactivation of the enzyme by H₂O₂. Comparison of the enzyme-linked immunosorbent assay method with microscopic and histochemical tests for the presence of the phony peach disease bacterium in 9-year-old “June Gold” peach trees gave comparable results. The peroxidase conjugate with the Trinder reagent is more sensitive than the alkaline phosphatase conjugate typically used for enzyme-linked immunosorbent assay quantitation.     

Relationship of Intracellular Coenzyme F420 Content to Growth and Metabolic Activity of Methanobacterium bryantii and Methanosarcina barkeri

The use of F₄₂₀ as a parameter for growth or metabolic activity of methanogenic bacteria was investigated. Two representative species of methanogens were grown in batch culture: Methanobacterium bryantii (strain M.o.H.G.) on H₂ and CO₂, and Methanosarcina barkeri (strain Fusaro) on methanol or acetate. The total intracellular content of coenzyme F₄₂₀ was followed by high-resolution fluorescence spectroscopy. F₄₂₀ concentration in M. bryantii ranged from 1.84 to 3.65 μmol · g of protein⁻¹; and in M. barkeri grown with methanol it ranged from 0.84 to 1.54 μmol · g⁻¹ depending on growth conditions. The content of F₄₂₀ in M. barkeri was influenced by a factor of 2 depending on the composition of the medium (minimal or complex) and by a factor of 3 to 4 depending on whether methanol or acetate was used as the carbon source. A comparison of F₄₂₀ content with protein, cell dry weight, optical density, and specific methane production rate showed that the intracellular content of F₄₂₀ approximately followed the increase in biomass in both strains. In contrast, no correlation was found between specific methane production rate and intracellular F₄₂₀ content. However, qCH₄(F₄₂₀), calculated by dividing the methane production rate by the coenzyme F₄₂₀ concentration, almost paralleled qCH₄(protein). These results suggest that F₄₂₀ may be used as a specific parameter for estimating the biomass, but not the metabolic activity, of methanogens; hence qCH₄(F₄₂₀) determined in mixed populations with complex carbon substrates must be considered as measure of the actual methanogenic activity and not as a measure of potential activity.     

Relationship of Intracellular Coenzyme F(420) Content to Growth and Metabolic Activity of Methanobacterium bryantii and Methanosarcina barkeri

The use of F(420) as a parameter for growth or metabolic activity of methanogenic bacteria was investigated. Two representative species of methanogens were grown in batch culture: Methanobacterium bryantii (strain M.o.H.G.) on H(2) and CO(2), and Methanosarcina barkeri (strain Fusaro) on methanol or acetate. The total intracellular content of coenzyme F(420) was followed by high-resolution fluorescence spectroscopy. F(420) concentration in M. bryantii ranged from 1.84 to 3.65 mumol . g of protein; and in M. barkeri grown with methanol it ranged from 0.84 to 1.54 mumol . g depending on growth conditions. The content of F(420) in M. barkeri was influenced by a factor of 2 depending on the composition of the medium (minimal or complex) and by a factor of 3 to 4 depending on whether methanol or acetate was used as the carbon source. A comparison of F(420) content with protein, cell dry weight, optical density, and specific methane production rate showed that the intracellular content of F(420) approximately followed the increase in biomass in both strains. In contrast, no correlation was found between specific methane production rate and intracellular F(420) content. However, qCH(4)(F(420)), calculated by dividing the methane production rate by the coenzyme F(420) concentration, almost paralleled qCH(4)(protein). These results suggest that F(420) may be used as a specific parameter for estimating the biomass, but not the metabolic activity, of methanogens; hence qCH(4)(F(420)) determined in mixed populations with complex carbon substrates must be considered as measure of the actual methanogenic activity and not as a measure of potential activity.     

Quantification of Dehalospirillum multivorans in Mixed-Culture Biofilms with an Enzyme-Linked Immunosorbent Assay

A fast, highly selective and sensitive method to quantify specific biomasses in mixed-culture biofilms is described. It consists of detachment of a biofilm from its support material, resolution of the detached biofilm flocs in order to separate the enclosed cells and antigens, and quantification of specific biomass by an enzyme-linked immunosorbent assay.     

Isolation and characterization of disaggregatase from Methanosarcina mazei LYC

At certain stages in its growth cycle, Methanosarcina mazei produces an enzyme (disaggregatase) that causes aggregates to separate into single cells. M. mazei S-6 and LYC both produce this enzymatic activity, although the specificities of activities differ. The disaggregatase of M. mazei S-6 had little effect on strain LYC cells, but the disaggregatase of M. mazei LYC disaggregated both strain LYC and strain S-6 cells. The disaggregatase of M. mazei LYC was purified by column chromatography, and it apparently consisted of two similar subunits with a combined molecular size of about 180,000 Da. Strain S-6 culture supernatants contained 14 U of activity per liter when activity was measured as uronic acids released from purified cell wall material. When the activity was quantified as the release of uronic acids from boiled M. mazei S-6 cells, the highest activity was found at pH 4.7 and at 35 degrees C.     

Ultrasound Treatment for Harvesting an Aminopeptidase from Lactic Acid Bacteria and Quantitation of the Enzyme by Enzyme-Linked Immunosorbent Assays

Ultrasound treatment of Lactococcus lactis subsp. cremoris AM2 was optimized to release a maximum amount of intracellular aminopeptidase without modifying the antigenicity of the enzyme. The cells were sonicated three times for 30 s at 23 W. Antibodies produced against the aminopeptidase purified from L. lactis subsp. cremoris AM2 enabled us to use immunoblotting to detect the enzyme in the lysates of all of the lactococci tested but not in the lysates of Leuconostoc strains, lactobacilli, and Streptococcus salivarus subsp. thermophilus. A sandwich enzyme-linked immunosorbent assay (ELISA) was developed to quantify the purified aminopeptidase; the detection limit was 4 ng/ml. The aminopeptidase in the supernatant obtained after the ultrasound treatment of strain AM2 cells was detected with the ELISA starting with a total protein concentration of 200 ng/ml. The proportion of equivalent purified aminopeptidase in the supernatant of L. lactis subsp. cremoris AM2 was about 2% of the total protein. Similarly, the aminopeptidase was quantified in different lactococci; the percentages varied between 0.16 and 2%, depending on the strain. The aminopeptidase content in a mixture of several lactic bacteria was also determined with the sandwich ELISA.     

Isolation and characterization of the iron-containing superoxide dismutase of Methanobacterium bryantii

Methanobacterium bryantii contains a single electrophoretically discernible superoxide dismutase, which constitutes 0.4% of the extractable protein. This enzyme has been purified to electrophoretic and ultracentrifugal homogeneity. It appears to be a tetramer. The subunits were tenaciously, but noncovalently bonded and were of identical size. The molecular weight of the enzyme was found to be 91,000 ± 2000. The specific activity of this enzyme was identical to that previously noted for the corresponding enzyme from Escherichia coli. The enzyme contained 2.7 atoms of Fe, 1.7 atoms of Zn, and less than 0.2 atoms Mn per tetramer. Its amino acid composition placed this enzyme with the other Mn- and Fe-containing superoxide dismutases. The M. bryantii enzyme was also similar to previously described Fe-containing superoxide dismutases in its optical and electron paramagnetic resonance spectra and in its susceptibility to inactivation by H₂O₂. The M. bryantii enzyme was ininhibited by N₃^?, but was less sensitive towards this inhibitor than other iron-containing superoxide dismutases.     

Enrichment Double-Antibody Sandwich Indirect Enzyme-Linked Immunosorbent Assay That Uses a Specific Monoclonal Antibody for Sensitive Detection of Ralstonia solanacearum in Asymptomatic Potato Tubers

Sensitive and specific routine detection of Ralstonia solanacearum in symptomless potato tubers was achieved by efficient enrichment followed by a reliable double-antibody sandwich indirect enzyme-linked immunosorbent assay based on the specific monoclonal antibody 8B-IVIA. This monoclonal antibody reacted with 168 typical R. solanacearum strains and did not recognize 174 other pathogenic or unidentified bacteria isolated from potato. The optimized protocol included an initial enrichment step consisting of shaking the samples in modified Wilbrink broth for 72 h at 29°C. This step enabled specific detection by the enzyme-linked immunosorbent assay of 1 to 10 CFU of R. solanacearum per ml of initial potato extract. Analysis of 233 commercial potato lots by this method provided results that coincided with the results of conventional methods.     

Spontaneous Disaggregation of Methanosarcina mazei S-6 and Its Use in the Development of Genetic Techniques for Methanosarcina spp

When monomethylamine was the growth substrate, spontaneous disaggregation of Methanosarcina mazei S-6 commenced at the mid-exponential phase and resulted in the formation of a suspension containing 10⁸ to 10⁹ free cells per ml. Free cells were osmotically fragile and amenable to extraction of DNA. Hypertonic media for the manipulation and regeneration of free cells into aggregates were developed, and plating efficiencies of 100% were achieved for M. mazei S-6 and LYC. Free cells of strain S-6 required MgCl₂ (10 mM) for growth, whereas aggregates did not. Specific growth rates of strains S-6 and LYC were increased by MgCl₂. Treatment with pronase caused sphere formation and removal of the protein wall of cells of strain S-6, but protoplasts could not be regenerated. The disaggregating enzyme produced by strain S-6 facilitated the preparation of suspensions of free cells of some strains of Methanosarcina barkeri. Although this provided a means of extracting high-molecular-weight DNA from M. barkeri, less than 0.1% of free cells were viable.     

Enzyme-Linked Immunosorbent Assay for Detection of Chitooligosaccharides

In order to detect chitooligosaccharides (COS), an enzyme-linked immunosorbent assay (ELISA) was developed. A chitooligosaccharide mixture (COSM) conjugated to bovine serum albumin was used to immunize rabbits to produce an anti-COS polyclonal antibody. By use of specific antibody and COSM-horseradish peroxidase conjugate, we established a competitive direct ELISA (cdELISA) the detection limit of which was about 0.1 μg/ml. In the cdELISA, the cross-reactivities of the specific antibody toward glucosamine, chitobiose, chitotriose, chitotetraose, chitopentaose, and chitohexaose were 0.27, 27, 75, 75, 144, and 100%, respectively, and those toward N-acetylchitobiose, N-acetylchitotriose, N-acetylchitotetraose, N-acetylchitopentaose, and N-acetylchitohexaose were 1.58, 0.005, 1.08, 0.05, and 0.40%, respectively.     

Isolation and characterization of disaggregatase from Methanosarcina mazei LYC.

At certain stages in its growth cycle, Methanosarcina mazei produces an enzyme (disaggregatase) that causes aggregates to separate into single cells. M. mazei S-6 and LYC both produce this enzymatic activity, although the specificities of activities differ. The disaggregatase of M. mazei S-6 had little effect on strain LYC cells, but the disaggregatase of M. mazei LYC disaggregated both strain LYC and strain S-6 cells. The disaggregatase of M. mazei LYC was purified by column chromatography, and it apparently consisted of two similar subunits with a combined molecular size of about 180,000 Da. Strain S-6 culture supernatants contained 14 U of activity per liter when activity was measured as uronic acids released from purified cell wall material. When the activity was quantified as the release of uronic acids from boiled M. mazei S-6 cells, the highest activity was found at pH 4.7 and at 35 degrees C.     

Comparison of PCR, Electrochemical Enzyme-Linked Immunosorbent Assays, and the Standard Culture Method for Detecting Salmonella in Meat Products

An electrochemical enzyme-linked immunosorbent assay (ELISA) coupled with flow injection analysis (ELISA-FIA) and a PCR-based method using ST11 and ST15 primers for detecting salmonellae in meat were evaluated in comparison with the International Organization for Standardization (ISO) culture method. The methods were applied to experimentally contaminated and naturally contaminated meat samples. The results showed that both ELISA-FIA and PCR allowed detection of salmonella in a product contaminated with a low number of the microorganisms (1 to 10 salmonellae/25 g) after only 5 h of incubation of preenrichment broth, and they were just as effective as the ISO method.     

Enzyme-Linked Immunosorbent Assay for Specific Identification and Enumeration of Azospirillum brasilense Cd. in Cereal Roots

The enzyme-linked immunosorbent assay is suggested as a reliable, sensitive, and highly specific method for the identification and enumeration of Azospirillum brasilense Cd. As few as 10⁵ CFU/ml can be practically identified by this method. At higher bacterial numbers, sensitivity increased linearly up to 5 × 10⁸ CFU/ml, yielding useful standard curves. No cross-reaction was found either with different closely related Azospirillum strains or with other rhizosphere bacteria. The method allows for a specific identification of A. brasilense Cd. both in pure cultures and in mixtures with other bacterial species, even when the colony morphology is variable. The method was successfully applied to assess the degree of root colonization on various cereals by A. brasilense Cd.     

Immunochemistry and localization of the enzyme disaggregatase in Methanosarcina mazei.

The enzyme disaggregatase (Dag) from Methanosarcina mazei was studied immunochemically. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified Dag under reducing and nonreducing conditions revealed a single band with a 94-kDa molecular mass. Dag was found to be immunogenic in rabbits; a polyclonal antibody probe was prepared and used to detect the enzyme by slide immunoenzymatic assay, immunofluorescence, and immunoblotting in various species of Methanosarcina known to convert from packets to single cells, including M. mazei. The enzyme could not be detected in other members of the family Methanosarcinaceae that do not convert. By immunogold electron microscopy, Dag was mapped to the cell wall of packets and to the cell membrane of single cells of two M. mazei strains.     

Activation of the methylreductase system from Methanobacterium bryantii by corrins.

Corrins activated the methylreductase system from Methanobacterium bryantii three- to fivefold in extracts resolved from low-molecular-weight factors. Corrins did not substitute for ATP and component B, which were also required for maximal activity. The concentration of diaquacobinamides required for one-half maximal activity was 1 microM. The concentrations of cyanocobalamin, methylcobalamin, Co alpha-(5-hydroxybenzimidazoyl)-Co beta-cyanocobamide, and 5'-deoxyadenosylcobinamide required for one-half maximal activity were between 4 and 7 microM. Deoxyadenosylcobalamin was nearly inactive. Activation was independent of thiols, coenzyme M, and ATP. Activation was also observed after partial purification of the methylreductase system by agarose column chromatography. Corrins were required in catalytic concentrations, methylcobalamin was not required, and methanogenesis was enzymatic. Corrin activation of the methylreductase is a novel effect on methanogenesis. However, the physiological significance of the corrin activation is uncertain.     

Activation of the methylreductase system from Methanobacterium bryantii by ATP.

The methylreductase of Methanobacterium bryantii required ATP for activity. There was sufficient ATP synthesis in extracts to account for the observed activity. Hexokinase inhibited the methylreductase by competing for endogenously synthesized ATP. The uncoupler, carbonyl cyanide p-trifluoromethyoxyphenyl hydrazone, inhibited only at concentrations greater than 0.5 mM, and detergents and non-halogenated membrane-permeable-ions did not inhibit. Thus, membrane proton gradients are not important in activation. In addition, maximal activation was obtained with less than 0.25 mM ATP, was inhibited by beta, gamma-imido ATP, and was strongly temperature dependent. The activated state was very unstable, having a half-life of 5 to 15 min. After gel filtration at 5 degrees C, the methylreductase retained partial activity for a short time in the absence of ATP. These observations indicate that activation involves the modification of a protein or protein-bound cofactor of the methylreductase system.     

Characterization of Egg Yolk Antibodies for Detection and Quantification of Selenomonas ruminantium by Using an Enzyme-Linked Immunosorbent Assay

The specificity of polyclonal antibodies prepared against strains of Selenomonas ruminantium, the effect of assay conditions, and quantification of individual strains in mixed-cell suspensions of selenomonad strains were examined in this study. Whole-cell suspensions were prepared with pure cultures of S. ruminantium PC18, HD₄, GA192, and D. Each cell suspension was injected into a Leghorn laying hen, and polyclonal antibodies were harvested from eggs laid in week 3 or 7 following initial immunization. Antibodies made to the S. ruminantium strains readily discerned the homologous strain from the heterologous strains. Cross-reactivity among antibodies and the heterologous S. ruminantium strains ranged from 5 to 26%. Among non-S. ruminantium species, cross-reactivity of S. ruminantium antibodies was greatest with Selenomonas sputigena (3 to 34%) and Succinivibrio dextrinosolvens (0 to 37%). Antibodies made to strains GA192 and D were used to quantify a mixture of the two strains. Both antibodies responded to graded concentrations of the homologous antigen in the biculture mixtures in accord with the change in the direct cell counts for each strain (strain D, R² = 0.92; strain GA192, R² = 0.90). This enzyme-linked immunosorbent assay enabled concurrent and accurate quantification of two strains of S. ruminantium subsp. ruminantium in a mixed-cell suspension with a precision of much less than 1 order of magnitude.     

Functional and structural characterization of the Methanosarcina mazei proteasome and PAN complexes

We have cloned the proteasome and the proteasome activating nucleotidase (PAN) genes from the mesophilic archaeon Methanosarcina mazei and produced the respective proteins in Escherichia coli cultures. The recombinant complexes were purified to homogeneity and characterized biochemically, structurally, and by mass spectrometry. We found that the degradation of Bodipy-casein by Methanosarcina proteasomes was activated by Methanosarcina PAN. Notably, the Methanosarcina PAN unfolded GFP-SsrA only in the presence of Methanosarcina proteasomes. Structural analysis by 2D averaging electron microscopy of negatively stained complexes displayed the typical structure for the proteasome, namely four-striped side-views and sevenfold-symmetric top-views, with 15 nm height and 11 nm diameter. The structural analysis of the PAN preparation revealed also four-striped side-views, albeit with a height of 18 nm and sixfold-symmetric top-views with a diameter of 15 nm, which corresponds most likely to a dimer of two hexameric complexes. Mass spectrometric analysis of both the Methanosarcina and the Methanocaldococcus PAN proteins indicated hexameric complexes. In summary, we performed a functional and structural characterization of the PAN and proteasome complexes from the archaeon M. mazei and described unique new structural and functional features.     

Enzyme-Linked Immunosorbent Assay for Detection of Ochratoxin A

A simple microtest plate enzyme-linked immunosorbent assay was developed for the detection of ochratoxin A at levels as low as 25 pg per assay. The relative cross-reactivities of the antibody in this system with ochratoxin A (OA), OB, OC, and Oα were found to be 1.0, 0.14, 0.44, and 0.01, respectively.