Endogenous GUS Activity Research Articles

Off-on fluorogenic substrate harnessing ESIPT and AIE features for in situ and long-term tracking of β-glucuronidase in Escherichia coli

Detection and isolation of both O157:H7 and non-O157:H7 Escherichia coli are essential for the evaluation and surveillance of the associated foodborne disease risk. However, the development of a chromogenic/fluorogenic culture medium for detection and isolation is challenging yet urgent. β-Glucuronidase (GUS) serves as an important biomarker in E. coli. A novel and simple off-on fluorogenic substrate (BTBP-Gluc) was developed, based on a solid-state fluorophore (BTBP) with excited-state intramolecular proton transfer (ESIPT) and aggregation-induced emission (AIE) effects. BTBP-Gluc could be well-suited for visually detecting GUS activity under acidic conditions (3.0 ≤ pH＜7.0). It was successfully applied for detection of endogenous intracellular GUS activity in living E. coli and could produce the in situ and long-term localization of the target colonies and cell imaging of living E. coli. Additionally, it showed high selectivity, little toxicity, excellent cell permeability and sensitivity. BTBP-Gluc was combined with two tightly localizing chromogenic substrates, Magenta-Gal and X-Glu. These combinations were applied to the development of a new chromogenic-fluorogenic culture medium (C–F E. coli agar). On this new agar simultaneous visual detection and isolation of O157:H7 and non-O157:H7 E. coli with relatively high accuracy was achieved. C–F E. coli agar shows potential in serving as a simple sensitive tool for detecting and isolating O157:H7 and non-O157:H7 E. coli in food samples such as milk.

English

We have chosen rice as a model crop to ascertain endogenous β-glucuronidase (family 79) activity and to differentiate the same from Escherichia coli based β-glucuronidase (family-2). The investigation dwells on characterizing endogenous β-glucuronidase (GUS) activity during the early stages of seed germination and from rice callus. Also, similar studies were made from homozygous transgenic rice line expressing E. coli GUS under the control of glyoxalase I promoter. Endogenous GUS activity was detected in plumules, shoots and calli of rice, nevertheless, showing differential response to pH. Further, endogenous GUS in rice was specifically inhibited by saccharic acid 1,4-lactone (SL) but, the E. coli β-glucuronidase remain unaffected, indicating distinct biochemical properties of family-2 and family-79 β-glucuronidase. Key words: Family 2, family 79, X-gluc, saccharic acid, 1,4-lactone.

Potential Repurposing of Known Drugs as Potent Bacterial β-Glucuronidase Inhibitors

The active metabolite of the chemotherapeutic irinotecan, SN-38, is detoxified through glucuronidation and then excreted into the gastrointestinal tract. Intestinal bacteria convert the glucuronidated metabolite back to the toxic SN-38 using β-glucuronidase (GUS), resulting in debilitating diarrhea. Inhibiting GUS activity may relieve this side effect of irinotecan. In this study, we sought to determine whether any known drugs have GUS inhibitory activity. We screened a library of Food and Drug Administration–approved drugs with a cell-free biochemical enzyme assay using purified bacterial GUS. After triage, five drugs were confirmed to inhibit purified bacterial GUS. Three of these were the monoamine oxidase inhibitors nialamide, isocarboxazid, and phenelzine with average IC50 values for inhibiting GUS of 71, 128, and 2300 nM, respectively. The tricyclic antidepressant amoxapine (IC50 = 388 nM) and the antimalarial mefloquine (IC50 = 1.2 µM) also had activity. Nialamide, isocarboxazid, and amoxapine had no significant activity against purified mammalian GUS but showed potent activity for inhibiting endogenous GUS activity in a cell-based assay using living intact Escherichia coli with average IC50 values of 17, 336, and 119 nM, respectively. Thus, nialamide, isocarboxazid, and amoxapine have potential to be repurposed as therapeutics to reduce diarrhea associated with irinotecan chemotherapy and warrant further investigation for this use.

Factor affecting the endogenous β-glucuronidase activity in rapeseed haploid cells: How to avoid interference with the Gus transgene in transformation studies

The gus gene is one of the most frequently used reporter genes in transgenic plants. However, this gene can only be used if the selected plant species does not show endogenous GUS activity. Rapeseed ( Brassica napus) microspores and microspore-derived embryos (MDEs) were found to exhibit high activity of endogenous β-glucuronidase which interferes with the expression of bacterial β-glucuronidase that was transferred into these tissues by biolistic transformation. In order to eliminate this background activity from rapeseed MDEs, different pHs of the assay buffer (5.8, 7 and 8) with or without methanol in the reaction buffer and incubation of these tissues at different temperatures (24 °C, 38 °C and 55 °C) were investigated. To avoid this problem in microspores, two incubation temperatures (38 °C and 55 °C) at different periods after GUS assay (4, 24 and 48 h) and in the presence of 1 mM potassium ferricyanide and 1 mM potassium ferrocyanide were tested. The endogenous GUS activity was significantly decreased in transformed and untransformed MDEs, when the phosphate buffer was adjusted to pH 8 and 28% methanol in the reaction solution was used. In rapeseed microspores, use of 1 mM potassium ferricyanide and 1 mM potassium ferrocyanide in the reaction buffer enhanced the expression rate of gus transgene rather than endogenous GUS activity where the high levels of gus transgene expression was observed 4 h after histochemical GUS assay. Incubation of rapeseed microspores and MDEs at 55 °C completely eliminated the endogenous GUS activity. In this study, we also examined changes in endogenous GUS activity in rapeseed MDEs at several stages including the globular, heart, torpedo and cotyledonary stages. The level of endogenous GUS activity was increased 4.33 folds in heart embryos, 6.54 folds in torpedo embryos and 8.5 folds in cotyledonary embryos. Furthermore, the level of GUS activity increased 1.72 folds in MDEs of B. napus in 12-h treatment with 2 μM gibberellic acid.

A novel method for fluorometric continuous measurement of β-glucuronidase (GUS) activity using 4-methyl-umbelliferyl-β- d-glucuronide (MUG) as substrate

β-Glucuronidases (GUS) are histochemically and fluorometrically detectable enzymes that cleave the glycosidic bond of glucuronides in living organisms. Previously thought to be absent in plants, endogenous GUS activity has recently been demonstrated ubiquitous, and its function is being widely investigated in model plants. Further, the GUS gene from Escherichia coli is the most widely used reporter gene in plant transformation experiments. Fluorometric assay of GUS activity is universally performed using fluorogenic substrate 4-methyl-umbelliferyl-β- d-glucuronide (MUG) by discontinuous measurement, based on the general notion that basification of the solution following enzymatic cleavage is necessary to detect the fluorophore 4-methyl-umbelliferone (MU). In this report, we analyze MU and MUG spectroscopic characteristics at different pHs, and show that MU is a fluorescent compound also at pHs below its p K a, although with a different excitation spectrum compared to the ionized form. On the basis of this evidence, we show that MUG is a suitable substrate to perform continuous measurement of GUS activity at the pH optima of plant endogenous and E. coli GUS enzymes. An efficient and straightforward method which greatly improves the procedure to assess GUS activity is described, and insights are given on the principles leading to optimal setting of instrumental parameters.

Purification, Cloning and Functional Characterization of an Endogenous beta-Glucuronidase in Arabidopsis thaliana

Beta-glucuronidase (GUS) activities have been extensively characterized in bacteria, fungi, and animals, and the bacterial enzyme GUSA from Escherichia coli is commonly used as a reporter for gene expression studies in plants. Although endogenous GUS activity has been observed in plants, the nature and function of the enzymes involved remain elusive. Here we report on tissue-specific localization, partial purification and identification of AtGUS2, a GUS active under acidic conditions from Arabidopsis thaliana. This enzyme belongs to the GH79 family in the Carbohydrate-Active Enzymes database, which also includes mammalian heparanases that degrade the carbohydrate moieties of cell surface proteoglycans, and fungal enzymes active on arabinogalactan proteins (AGPs). We characterized a knockout insertion line (atgus2-1) and transgenic lines overexpressing AtGUS2 (Pro(35S):AtGUS2). Endogenous GUS activity assayed histochemically and biochemically was absent in atgus2-1 tissues and four times higher in Pro(35S):AtGUS2 lines. AGPs purified from atgus2-1 and Pro(35S):AtGUS2 seedlings showed higher and markedly lower glucuronic acid content, respectively. Our results suggest that endogenous GUS activity influences the sugar composition of the complex polysaccharide chains of AGPs. We also show that transgenics display hypocotyl and root growth defects compared to wild-type plants. Hypocotyl and root lengths are increased in Pro(35S):AtGUS2 seedlings, whereas hypocoyl length is reduced in atgus2-1 seedlings. These data are consistent with a role for the carbohydrate moieties of AGPs in cell growth.

Characterization of pedicel β‐glucuronidase activity in developing maize (Zea mays) kernels

A conspicuous endogenous maize (Zea mays L.) β‐glucuronidase (GUS) activity was observed in histochemical assays of non‐transformed maize kernels, confounding the use of Escherichia coli gusA as a reporter gene. Appearance of the endogenous activity was developmentally dependent and highly tissue‐specific, being localized to the upper pedicel (basal maternal kernel) tissues where the black layer forms in the latter stages of kernel development. Pedicel homogenates exhibited GUS activity using either p‐nitrophenyl‐β‐D‐glucuronide or 4‐methylumbelliferyl‐β‐D‐glucuronide (MUG) as substrates. Pedicel GUS was apparently not the result of endophyte contamination of enzyme isolates since no endophytes could be cultured. The MUG‐based activity had a pH optimum of 4 to 5 and was separable into two isoforms by anion exchange chromatography with Km values for MUG of 2.2 and 2.7 µM for the early‐ and late‐eluting forms, respectively. The pedicel GUS isoforms had very similar characteristics: native Mr of approximately 32000, stimulation by assay at 60°C, inhibition at high ionic strength or in the presence of EDTA and relative insensitivity to the E. coli GUS inhibitor saccharic acid‐1,4‐lactone. Only the early‐eluting form, however, was capable of hydrolyzing the histocbemical GUS substrate 5‐bromo‐4‐chloro‐3‐indoyl‐β‐D‐glucuronide. Neither isoform exhibited antifungal activity against Fusarium moniliforme. In contrast to the in vitro activity, pedicel endogenous GUS measured histochemically was completely inhibited by saccharic acid‐1,4‐lactone, unaffected by EDTA and greatly decreased by incubation at elevated assay temperature. A modification of the standard histochemical GUS assay allowed complete suppression of endogenous GUS activity while enhancing E. coli‐derived GUS activity in kernels transiently expressing the gusA gene. Possible roles of these endogenous GUS activities within the black layer region of the kernel pedicel are proposed.

β-Glucuronidase activity in transgenic and non-transgenic tobacco cells: specific elimination of plant inhibitors and minimization of endogenous GUS background

Bacterial β-glucuronidase is often introduced into plants as a reporter gene fused to constitutive or inducible promoters. However, the presence of both endogenous inhibitors of GUS activity and endogenous GUS enzymes in transgenic plants could lead to an underestimation of GUS. In this paper, a decrease of the V m values and a greater affinity ( K m) of the GUS enzyme for its substrate (p-NPG) has been recorded when increasing amounts of protein from untransformed tobacco cells has been added to the pure β-glucuronidase. The observed inhibition is not competitive and can be completely removed when the tobacco extracts are passed through Sephadex G-25 spin columns prior to the assays. After such a treatment, the activity of E. coli GUS in transgenic tobacco cells (constitutive or inducible systems) was stimulated by a factor 1.2 or 2 for p-NPG or 4-MUG substrates, respectively. This method was also effective in suppressing the endogenous GUS or GUS-like activity which can interfere with the activity originating from the introduced GUS gene.

Methanol does not specifically inhibit endogenous β-glucuronidase (GUS) activity

One of the problems associated with the use of the bacterial gus A gene as a reporter is the presence of ‘endogenous GUS’ activity in some plant species. A recent paper [1] suggests that the addition of methanol to GUS reaction buffer specifically inhibits endogenous GUS activity while enhancing the activity of an introduced gus A gene. The results described in this paper suggest this phenomenon may be explained by the effect methanol has on cell membranes and demonstrate that it does not specifically inhibit endogenous GUS activity.

DNA Methylation Is Involved in Maintenance of an Unusual Expression Pattern of an Introduced Gene

In one of 30 transgenic tobacco (Nicotiana tabacum) plants, the expression of an introduced beta-glucuronidase (GUS) gene driven by the cauliflower mosaic virus 35S promoter, was found to be repressed as the plant matured, whereas the endogenous GUS activity was unaffected. Plants grown from seeds or regenerated from leaf discs derived from this plant showed a similar temporal pattern of expression. Suspension-cultured cells established from nonexpressing leaves did not express the introduced gene. In these cells, the silent gene could be reactivated by treatment for 5 or 10 days with 5-azacytidine. Overall, demethylation of the genome preceded recovery of the enzyme activity. The increase in the fraction of reactivated cells progressed in two phases. Up to 8 weeks after starting the 5-azacytidine treatment, approximately 2 to 4% of the cells were expressing GUS, followed by a dramatic increase of GUS-expressing cells. Thirteen weeks after starting the 5-azacytidine treatment, the fraction of GUS-expressing cells amounted to 80%. At this time, the original overall level of DNA methylation was reestablished. The degree of DNA demethylation, as well as the magnitude of reactivation, was dependent on the duration of the 5-azacytidine treatment. These results demonstrate that DNA methylation appears to be involved in the regulation of the introduced GUS gene and that this development-dependent pattern of expression can be inherited.

Agrobacterium‐Mediated Transfer of the GUS Gene into Pollen of Petunia

AbstractTransfer of the GUS gene into pollen has been studied in co‐cultures of Agrobacterium and Petunia pollen. The Agrobacterium strain used contains a GUS gene between the two borders of the T‐DNA. Uptake and integration of this GUS gene could be shown using two different restriction systems. First, by appropriate cleavage within the T‐DNA the GUS gene could be isolated intact from Agrobacterium‐treated pollen. Second, using enzymes with cleave the T‐DNA only once, integration of this T‐DNA into individual pollen genomes could be shown. The fragments obtained could not be obtained from Agrobacterium alone. The positive Southern blots were reprobed with vir probes, but all were negative. Also following plating, no Agrobacteria could be detected from our pollen DNA preparations. Therefore, the signals obtained were not due to contaminating bacteria. Due to a high endogenous GUS activity of Petunia pollen the expression of the transferred gene could not be studied. The data demonstrate the uptake and integration of T‐DNA into pollen and are closing a gap in the line of evidence for the functioning of an indirect Agrobacterium — mediated gene transfer. Besides this, it should be stressed that only this indirect pollen system leads to success.

An improved assay for β-glucuronidase in transformed cells: Methanol almost completely suppresses a putative endogenous β-glucuronidase activity

The bacterial β-glucuronidase (GUS) gene is often introduced into plants as a reporter gene fused to a promoter because of advantages over other reporter genes. However, many plants have non-neglegible levels of endogenous GUS or GUS-like activity which can interfere with the activity originating from the introduced GUS gene, especially if this level is a low one. To eliminate the endogenous GUS activity, we studied the effect of some water-soluble organic solvents on decreasing it and found that addition of methanol at 20% volume to a GUS reaction mixture was very effective, lowering the endogenous activity to 0–15% of the origin in all plant extracts tested. Moreover, methanol at this concentration enhanced by 1.4-fold the activity originating from the introduced GUS gene or GUS purified from E. coli. This method enabled us to determine GUS gene expression more reliably, after introduction of GUS gene fusion into rice protoplasts. In histochemical GUS analysis of transgenic tobacco, this method was also effective for suppressing the endogenous GUS activity.