Synthesis and evaluation of novel fluorogenic substrates for the detection of bacterial beta-galactosidase

AIMS: A widely used coumarin derivative is 7-hydroxy-4-methylcoumarin-beta-D-galactoside (4-methylumbelliferone-beta-D-galactoside; 4-MU-GAL). This galactoside is utilized as a substrate for the detection of the beta-galactosidase activity of coliform bacteria in water analysis. The intense fluorescence of coumarin-based molecules has enabled them to be incorporated into enzyme-based tests for the quantitative assay of indicator bacteria. The aim of this present study was to evaluate the potential of other coumarin derivatives, by synthesis of a selection of core coumarin molecules. METHODS AND RESULTS: Several coumarin derivatives were found to be more promising than 4-MU, with ethyl-7-hydroxycoumarin-3-carboxylate (EHC) giving a combination of greater fluorescence over a broad pH range and reduced growth inhibition with 12 representative coliform strains. On conversion to a beta-galactoside derivative, EHC-GAL generated a more rapid fluorescence than any other tested substrate. CONCLUSIONS: When tested in a broth assay format, based on most probable number (MPN), low numbers of coliforms were detected with EHC-GAL around 1 h earlier than with 4-MU-GAL. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study suggests that EHC-GAL should be evaluated as a substrate for the detection of coliforms in water analysis, due to a combination of the following favourable features: (i) reduced toxicity; (ii) increased fluorescence; (iii) pH stability of fluorescence; and (iv) rapid detection.     

Evaluation of p-naphtholbenzein-beta-D-galactoside as a substrate for bacterial beta-galactosidase

We describe the synthesis of a new substrate for the detection of beta-galactosidase and evaluate its performance in comparison with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) and cyclohexenoesculetinbeta-D-galactoside (CHE-Gal). Of 206 Enterobacteriaceae strains able to hydrolyze X-Gal, 194 (94.2%) hydrolyzed CHE-Gal and 192 (93.2%) hydrolyzed p-naphtholbenzein-beta-D-galactoside (PNB-Gal). We conclude that PNB-Gal is an effective substrate for the detection of beta-galactosidase.     

Evaluation of new chromogenic substrates for the detection of coliforms

Aims: To evaluate a new range of chromogenic substrates for the detection of β‐galactosidase activity in coliforms and to compare their performance in agar media and broths. Methods and Results: Sixteen novel galactoside substrates were prepared and incorporated into agar and broth. Their performance was compared using Escherichia coli (five strains), Salmonella (two strains), Enterobacter (two strains), Klebsiella, Pseudomonas, Listeria, Serratia, Shigella, Citrobacter, Proteus and Staphylococcus as well as pathological urine samples. The six substrates out of the initial 16 that showed the greatest sensitivity were VQE‐gal, VQM‐gal, VLPr‐gal, VLE‐gal, VLM‐gal and VBzTM‐gal, whose released chromophores were red, brown or purple. VQE‐gal and VLPr‐gal were studied in greater detail and were incorporated into agar medium. Coliform colonies appeared red and brown respectively, following incubation at 37°C for 24 h; however, positive results were obtained within a working day. The VQE‐gal medium was compared with some commercially available media. Conclusions: The range of substrates described can be used in broths as well as in agars. The VQE agar allows the detection of coliforms within a working day. VQE‐gal medium proved to be more sensitive when compared to other available chromogenic media and allows the unambiguous detection of coliforms.     

Use of fluorescein-di-beta-D-galactopyranoside (FDG) and C12-FDG as substrates for beta-galactosidase detection by flow cytometry in animal, bacterial, and yeast cells.

Fluorescein-di-beta-D-galactopyranoside (FDG) was found to be a useful substrate for beta-galactosidase detection by flow cytometry in gram-negative bacteria, since it entered viable cells and gave a fluorescence emission proportional to the enzymatic activity. C12-FDG, a more lipophilic derivative, gave a very poor signal because of the lack of penetration. On the contrary, C12-FDG was more sensitive than FDG for beta-galactosidase activity determinations in animal cells. In contrast to previous reports, C12-FDG did not enter viable yeast cells, so that the use of the substrate required cell permeabilization. Without this treatment, C12-FDG penetrates only nonviable yeast cells that may occur in populations expressing beta-galactosidase.     

Activity of human hepatic beta-galactosidase toward natural glycosphingolipid substrates.

1. Human hepatic "acid" beta-galactosidase preparations, which had been purified approximately 250-fold, were examined for activities toward 4-methylumbelliferyl beta-galactoside, galactosylceramide, lactosylceramide, galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosyl-glucosylceramide (GM1-Ganglioside) and galactosyl-Cacetylgalactosaminyl-galactosyl-glucosylceramide (asialo GM1-ganglioside). 2. The enzyme was active toward the synthetic substrate, GM1-ganglioside and asialo GM1-ganglioside but was inactive toward galactosylceramide. Under our assay conditions, optimized for lactosylceramidase II, the preparations were as active toward lactosylceramide as toward GM1-ganglioside or its asialo derivative. Teh apparent Km values for the three natural substrates were similar. When determined by the assay system of Wenger, D.A., Sattler, M., Clark, C. and McKelvey, H. (1974) Clin. Chim. Acta 56, 199-206, lactosylceramidecleaving activity was 0.2% of that determined by our assay system. This confirmed our previous suggestion that the Wenger assay system determines exclusively the activity of lactosylceramidase I, which is probably identical with galactosylceramide beta-galactosidase. 3. Crude sodium taurocholate was far more effective than pure taurocholate in stimualting hydrolysis of the three glycosphingolipids by the beta-galactosidase. However, crude tauroxycholate, suggesting that the unique activating capacity of the crude taurocholate might be due to taurodeoxycholate present as the major impurity. 4. Cl- was generally stimulatory for hydrolysis of the natural glycosphingolipids by our enzyme preparation. Effects of additional oleic acid and Triton X-100 Were generally minor in either direction. 5. When the enzyme preparation was diluted with water, activity toward the synthetic substrate declined rapidly while those toward the natural substrates were essentially stable. Activity toward the synthetic substrate remained much more stable when the enzyme was diluted with 0.1 M sodium citrate/phosphate buffer, pH 5.0. 6. These observations provide insight into the complex relationship among the human hepatic beta-galactosidases.     

Evaluation of novel chromogenic substrates for the detection of bacterial beta-glucosidase

AIMS: To evaluate three previously unreported substrates for the detection of beta-glucosidase activity in clinically relevant bacteria and to compare their performance with a range of known substrates in an agar medium. METHODS AND RESULTS: The performance of 11 chromogenic beta-glucosidase substrates was compared using 109 Enterobacteriaceae strains, 40 enterococci and 20 strains of Listeria spp. Three previously unreported beta-glucosides were tested including derivatives of alizarin, 3',4'-dihydroxyflavone and 3-hydroxyflavone. These were compared with esculin and beta-glucoside derivatives of 3,4-cyclohexenoesculetin, 8-hydroxyquinoline and five indoxylics. All substrates yielded coloured precipitates upon hydrolysis in agar. Alizarin-beta-D-glucoside was the most sensitive substrate tested and detected beta-glucosidase activity in 72% of Enterobacteriaceae strains and all enterococci and Listeria spp. The two flavone derivatives showed poor sensitivity with Gram-negative bacteria but excellent sensitivity with enterococci and Listeria spp. CONCLUSIONS: Alizarin-beta-d-glucoside is a highly sensitive substrate for detection of bacterial beta-glucosidase and compares favourably with existing substrates. beta-glucosides of 3',4'-dihydroxyflavone and 3-hydroxyflavone are effective substrates for the detection of beta-glucosidase in enterococci and Listeria spp. SIGNIFICANCE AND IMPACT OF THE STUDY: The data presented allow for informed decisions to be made regarding the optimal choice of beta-glucosidase substrate for detection of pathogenic and/or indicator bacteria.     

A sensitive method for the detection of beta-galactosidase in transfected mammalian cells.

A sensitive method has been developed for the detection of E. coli beta-galactosidase in transfected HeLa cells. The chromogenic substrate, CPRG (chlorophenol red-beta-D-galactopyranoside), was compared with ONPG (o-nitrophenyl-beta-D-galactopyranoside) by kinetic analysis with purified beta-galactosidase. The Km for CPRG was 1.35 mM and the Vmax was 21.4, whereas the Km for ONPG was 2.42 and the Vmax was 41.1. CPRG at 8.0 mM (6-fold Km) gave 86% of the Vmax and was used as the standard concentration for quantitation of enzyme levels. The Vmax for CPRG was half that for ONPG, and chlorophenol red has an extinction coefficient that is 21-fold higher than o-nitrophenol; these factors make CPRG about 10-fold greater in sensitivity for the quantitation of enzyme levels. The use of Nonidet P-40 to lyse the cells and the use of CPRG as substrate permitted the rapid detection of low levels of enzyme production from transfected human cells that could not be detected using ONPG.     

Evaluation of novel fluorogenic substrates for the detection of glycosidases in Escherichia coli and enterococci

AIMS: Enzyme substrates based on 4-methylumbelliferone are widely used for the detection of Escherichia coli and enterococci in water, by detection of beta-glucuronidase and beta-glucosidase activity respectively. This study aimed to synthesize and evaluate novel umbelliferone-based substrates with improved sensitivity for these two enzymes. METHODS AND RESULTS: A novel beta-glucuronide derivative based on 6-chloro-4-methylumbelliferone (CMUG) was synthesized and compared with 4-methylumbelliferyl-beta-D-glucuronide (MUG) using 42 strains of E. coli in a modified membrane lauryl sulfate broth. Over 7 h of incubation, the fluorescence generated from the hydrolysis of CMUG by E. coli was over twice that from MUG, and all of the 38 glucuronidase-positive strains generated a higher fluorescence with CMUG compared with MUG. Neither substrate caused inhibition of bacterial growth in any of the tested strains. Four beta-glucosidase substrates were also synthesized and evaluated in comparison with 4-methylumbelliferyl-beta-D-glucoside (MU-GLU) using 42 strains of enterococci in glucose azide broth. The four substrates comprised beta-glucoside derivatives of umbelliferone-3-carboxylic acid and its methyl, ethyl and benzyl esters. Glucosides of the methyl, ethyl and benzyl esters of umbelliferone-3-carboxylic acid, were found to be superior to MU-GLU for the detection of enterococci, especially after 18 h of incubation, while umbelliferone-3-carboxylic acid-beta-D-glucoside was inferior. However, the variability in detectable beta-glucosidase activity among the different strains of enterococci in short-term assays using the three carboxylate esters (7 h incubation) may compromise their use for rapid detection and enumeration of these faecal indicator bacteria. CONCLUSIONS: The beta-glucuronidase substrate CMUG appears to be a more promising detection system than the various beta-glucosidase substrates tested. SIGNIFICANCE AND IMPACT OF THE STUDY: The novel substrate CMUG showed enhanced sensitivity for the detection of beta-glucuronidase-producing bacteria such as E. coli, with a clear potential for application in rapid assays for the detection of this indicator organism in natural water and other environmental samples.     

Alizarin-beta-D-galactoside: a new substrate for the detection of bacterial beta-galactosidase

We describe the synthesis of a new substrate for the detection of bacterial beta-galactosidase. This substrate, alizarin-beta-D-galactoside, is readily hydrolysed to release alizarin which complexes with various metal ions to form brightly coloured chelates. A total of 367 strains of Gram-negative bacteria were examined for their ability to hydrolyse three chromogenic substrates: alizarin-beta-D-galactoside (Aliz-gal), cyclohexenoesculetin-beta-D-galactoside (CHE-gal) and 5-bromo-4-chloro-3-indolyl-beta-D-galactoside (X-gal). A total of 182 strains (49.6%) were found to hydrolyse at least one of the three substrates. All of these 182 strains (100%) hydrolysed Aliz-gal whereas only 170 (93.4%) and 173 (95.1%) hydrolysed CHE-gal and X-gal, respectively. We conclude that alizarin-beta-D-galactoside is a highly sensitive substrate for the demonstration of beta-galactosidase.     

N-hydroxy peptides as substrates for alpha-chymotrypsin.

An N-hydroxylated peptide bond was found to be cleaved faster by an endopeptidase than the corresponding peptide bond. This preferred enzymatic cleavage was detected during proteolytic studies of the N-hydroxy peptide SIINFpsi[CO-N(OH)]GKL in the presence of the serine protease alpha-chymotrypsin in comparison with the natural SIINFEKL epitope and related analogs. For the first time, the replacement of the peptide bond by another motif afforded an oligomer which is degraded faster than the natural peptide. The N-hydroxy peptide is also more sensitive to the enzymatic degradation than the Gly-containing analog SIINFGKL. A tentative explanation for the unexpected higher cleavage rate of the CO-N(OH) bond is given on the basis of the N-OH intramolecular H-bonding capacity as indicated by NMR experiments. This property of the hydroxamate group may be of particular advantage for the introduction of a specific cleavage site within peptidomimetics or in prodrugs.     

Co-production of alpha-amylase and beta-galactosidase by Bacillus subtilis in complex organic substrates

Various nutrients belonging to three categories, carbon, organic nitrogen and complex organic sources, were investigated for the first time in terms of their effect on the co-production of extracellular thermostable alpha-amylase and beta-galactosidase by Bacillus subtilis, a bacterium isolated from fresh sheep's milk. Among the organic nitrogen sources tested, tryptone and corn steep liquor favored their production. Substitution of soluble starch by various starchy substrates, such as corn flour, had a positive effect on both enzyme yields. Furthermore, a two-fold higher production of both enzymes was achieved when corn steep liquor or tryptone was used in combination with the different flours. Among the divalent cations examined, calcium ions appeared to be vital for alpha-amylase production. The crude alpha-amylase and beta-galactosidase produced by this B. subtilis strain exhibited maximal activities at 135 degrees C and 65 degrees C, respectively, and were also found to be significantly stable at elevated temperatures.     

Fluorogenic substrates for the detection of microbial nitroreductases

AIMS: To synthesize and evaluate fluorogenic substrates for the detection of microbial nitroreductases. These substrates, all based on 7-nitrocoumarin, may be reduced to form fluorescent aminocoumarins. METHODS AND RESULTS: Thirty pathogenic microbial strains, including both bacteria and yeasts, were examined for nitroreductase activity in a whole-cell assay. All strains readily reduced each of the seven substrates to generate fluorescence, suggesting the widespread presence of nitroreductase activity in pathogenic bacteria. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: These novel substrates facilitate the direct detection of nitroreductase activity and have potential as sensitive indicators of microbial growth.     

Evaluation of procedures for reliable PCR detection of Ralstonia solanacearum in common natural substrates

Several procedures were compared for reliable PCR detection of Ralstonia solanacearum in common substrates (plant, seed, water and soil). In order to prevent the inhibition of PCR by substances contained in crude extracts, numerous DNA extraction procedures as well as additives to buffers or PCR mixtures were checked. Our results showed that the efficiency of these methods or compounds depended greatly upon the nature of the sample. Consequently, preparation of samples prior to PCR depended upon sample origin. Simple methods such as a combined PVPP/BSA treatment or the association of filtration and centrifugation for detecting the bacterium in plant or water samples were very powerful. DNA capture also efficiently overcame PCR inhibition problems and ensured the detection of R. solanacearum in environmental samples. However, the commercial DNA extraction QIAamp kit appeared to be the most effective tool to guarantee the accurate PCR detection of the pathogen whatever the origin of the sample; this was particularly true for soil samples where the commonly used methods for the detection of R. solanacearum were inefficient. This study demonstrates that using an appropriate procedure, PCR is a useful and powerful tool for detecting low levels of R. solanacearum populations in their natural habitats.     

A simple assay for DNA transfection by incubation of the cells in culture dishes with substrates for beta-galactosidase

Transfection efficiency of different cell types as well as promoter strength of cloned genes can be easily determined by direct assay of beta-galactosidase activity encoded from recombinant genes containing the E. coli beta-galactosidase gene. A substrate for beta-galactosidase, o-nitrophenyl-beta-D-galactopyranoside (ONPG), can be added to dishes containing the transfected cells, and the intensity of the colored enzyme product released from either the intact cell or cells lysed in the dishes can be determined. The results obtained by this assay are a reliable measure of transfection efficiency as well as promotor strength of the genes introduced into the cells. In addition, cells expressing the transfected gene can be identified and quantitated under a light microscope after incubation with X-gal. Thus, it is more convenient to use the E. coli beta-galactosidase gene than the chloramphenicol acetyltransferase gene as a reporter gene in the evaluation of DNA transfection.     

RNA tetraloops as minimalist substrates for aminoacylation.

Previous work established that seven-base-pair hairpin microhelices with sequences based on the acceptor stems of alanine, glycine, methionine, and histidine tRNAs can be aminoacylated specifically with their cognate amino acids. To obtain "minimalist" substrates with fewer base pairs, we took advantage of the high thermodynamic stability of RNA tetraloop motifs that are found in ribosomal RNAs. We show here that rationally designed RNA tetraloops with as few as four base pairs are substrates for aminoacylation. Major nucleotide determinants for recognition by the class II synthetases were incorporated into each of the respective tetraloop substrates, resulting in specific aminoacylation by the alanine, glycine, and histidine tRNA synthetases. An analysis of the kinetics of aminoacylation shows that, for the alanine system, the majority of the transition-state stabilization provided by the synthetase-tRNA interaction is reproduced by the interaction of the synthetase with nucleotides in its minimalist tetraloop substrate. In an extension of this work, we also observed specific aminoacylation with the class I methionine tRNA synthetase of RNA tetraloops based on sequences in the acceptor stem of methionine tRNA. Thus, the results demonstrate four different examples where specific aminoacylation is directed by sequences/structures contained in less than half of a turn of an RNA helix.     

Indigogenic substrates for detection and localization of enzymes

Indoxyl esters and glycosides are useful chromogenic substrates for detecting enzyme activities in histochemistry, biochemistry and bacteriology. The chemical reactions exploited in the laboratory are similar to those that generate indigoid dyes from indoxyl-beta-d-glucoside and isatans (in certain plants), indoxyl sulfate (in urine), and 6-bromo-2-S-methylindoxyl sulfate (in certain molluscs). Pairs of indoxyl molecules released from these precursors react rapidly with oxygen to yield insoluble blue indigo (or purple 6,6'-dibromoindigo) and smaller amounts of other indigoid dyes. Our understanding of indigogenic substrates was developed from studies of the hydrolysis of variously substituted indoxyl acetates for use in enzyme histochemistry. The smallest dye particles, with least diffusion from the sites of hydrolysis, are obtained from 5-bromo-, 5-bromo-6-chloro- and 5-bromo-4-chloroindoxyl acetates, especially the last of these three. Oxidation of the diffusible indoxyls to insoluble indigoid dyes must occur rapidly. This is achieved with atmospheric oxygen and an equimolar mixture of K(3)Fe(CN)(6) and K(4)Fe(CN)(6), which has a catalytic function. H(2)O(2) is a by-product of the oxidation of indoxyl by oxygen. In the absence of a catalyst, the indoxyl diffuses and is oxidized by H(2)O(2) (catalyzed by peroxidase-like proteins) in sites different from those of the esterase activity. The concentration of K(3)Fe(CN)(6)/K(4)Fe(CN)(6) in a histochemical medium should be as low as possible because this mixture inhibits some enzymes and also promotes parallel formation from the indoxyl of soluble yellow oxidation products. The identities and positions of halogen substituents in the indoxyl moiety of a substrate determine the color and the physical properties of the resulting indigoid dye. The principles of indigogenic histochemistry learned from the study of esterases are applicable to methods for localization of other enzymes, because all indoxyl substrates release the same type of chromogenic product. Substrates are commercially available for a wide range of carboxylic esterases, phosphatases, phosphodiesterases, aryl sulfatase and several glycosidases. Indigogenic methods for carboxylic esterases have low substrate specificity and are used in conjunction with specific inhibitors of different enzymes of the group. Indigogenic methods for acid and alkaline phosphatases, phosphodiesterases and aryl sulfatase generally have been unsatisfactory; other histochemical techniques are preferred for these enzymes. Indigogenic methods are widely used, however, for glycosidases. The technique for beta-galactosidase activity, using 5-bromo-4-chloroindoxyl-beta-galactoside (X-gal) is applied to microbial cultures, cell cultures and tissues that contain the reporter gene lac-z derived from E. coli. This bacterial enzyme has a higher pH optimum than the lysosomal beta-galactosidase of animal cells. In plants, the preferred reporter gene is gus, which encodes beta-glucuronidase activity and is also demonstrable by indigogenic histochemistry. Indoxyl substrates also are used to localize enzyme activities in non-indigogenic techniques. In indoxyl-azo methods, the released indoxyl couples with a diazonium salt to form an azo dye. In indoxyl-tetrazolium methods, the oxidizing agent is a tetrazolium salt, which is reduced by the indoxyl to an insoluble coloured formazan. Indoxyl-tetrazolium methods operate only at high pH; the method for alkaline phosphatase is used extensively to detect this enzyme as a label in immunohistochemistry and in Western blots. The insolubility of indigoid dyes in water limits the use of indigogenic substrates in biochemical assays for enzymes, but the intermediate indoxyl and leucoindigo compounds are strongly fluorescent, and this property is exploited in a variety of sensitive assays for hydrolases. The most commonly used substrates for this purpose are glycosides and carboxylic and phosphate esters of N-methylindoxyl. Indigogenic enzyme substrates are among many chromogenic reagents used to facilitate the identification of cultured bacteria. An indoxyl substrate must be transported into the organisms by a permease to detect intracellular enzymes, as in the blue/white test for recognizing E. coli colonies that do or do not express the lac-z gene. Secreted enzymes are detected by substrate-impregnated disks or strips applied to the surfaces of cultures. Such devices often include several reagents, including indigogenic substrates for esterases, glycosidases and DNAse.     

Detecting lacZ gene expression in living cells with new lipophilic, fluorogenic beta-galactosidase substrates.

Current methods for detecting lacZ expression in transformed cells are limited because they require such harsh conditions that viability of the cells after detection is drastically reduced. To overcome this problem, we developed a series of new substrates for detection of lacZ expression in living cells under standard culture or physiological conditions. After incubation with these fluorogenic substrates, cultured lacZ-positive mammalian cells appear morphologically normal, continue to divide, and retain the fluorescent product. Because the product is so well retained, fluorescence intensity can be quantitatively related to the level of gene expression. We have demonstrated this correlation using transformed yeast cells bearing various plasmids, each containing the lacZ gene and a unique promoter sequence with known capabilities for promoting gene expression in yeast.     

Lipid and steroid hydroperoxides as substrates for the non-selenium-dependent glutathione peroxidase.

The reduction of linoleic acid hydroperoxide catalysed by rat liver cytosol was previously shown to be catalysed by a selenium-dependent glutathione peroxidase. In contrast, the enzyme responsible in guinea-pig liver cytosol is not selenium-dependent and appears to be a glutathione transferase.