Effects of Global and Specific DNA-Binding Proteins on Transcriptional Regulation of the E. coli bgl Operon.

Using reporter gene (lacZ) transcriptional fusions, we examined the transcriptional dependencies of the bgl promoter (Pbgl) and the entire operon regulatory region (Pbgl-bglG) on eight transcription factors as well as the inducer, salicin, and an IS5 insertion upstream of Pbgl. Crp-cAMP is the primary activator of both Pbgl and the bgl operon, while H-NS is a strong dominant operon repressor but only a weak repressor of Pbgl. H-NS may exert its repressive effect by looping the DNA at two binding sites. StpA is a relatively weak repressor in the absence of H-NS, while Fis also has a weak repressive effect. Salicin has no effect on Pbgl activity but causes a 30-fold induction of bgl operon expression. Induction depends on the activity of the BglF transporter/kinase. IS5 insertion has only a moderate effect on Pbgl but causes a much greater activation of the bgl operon expression by preventing the full repressive effects of H-NS and StpA. While several other transcription factors (BglJ, RcsB, and LeuO) have been reported to influence bgl operon transcription when overexpressed, they had little or no effect when present at wild type levels. These results indicate the important transcriptional regulatory mechanisms operative on the bgl operon in E. coli.

Catabolism of aromatic ß-glucosides by bacteria can lead to antibiotics resistance.

Antimicrobial resistance is a serious public health threat worldwide today. Escherichia coli is known to resist low doses of antibiotics in the presence of sodium salicylate and related compounds by mounting non-heritable transient phenotypic antibiotic resistance (PAR). In the present study, we demonstrate that Bgl+ bacterial strains harboring a functional copy of the ß-glucoside (bgl) operon and are actively hydrolyzing plant-derived aromatic ß-glucosides such as salicin show PAR to low doses of antibiotics. The aglycone released during metabolism of aromatic ß-glucosides is responsible for conferring this phenotype by de-repressing the multiple antibiotics resistance (mar) operon. We also show that prolonged exposure of Bgl+ bacteria to aromatic ß-glucosides in the presence of sub-lethal doses of antibiotics can lead to a significant increase in the frequency of mutants that show heritable resistance to higher doses of antibiotics. Although heritable drug resistance in many cases is known to reduce the fitness of the carrier strain, we did not see a cost associated with resistance in the mutants, most of which carry clinically relevant mutations. These findings indicate that the presence of the activated form of the bgl operon in the genome facilitates the survival of bacteria in environments in which both aromatic ß-glucosides and antibiotics are present.

Effects of toxic ß-glucosides on carbohydrate metabolism in cotton bollworm, Helicoverpa armigera (Hübner).

The purpose of this study was to evaluate the effects of three toxic ß-glucosides, phlorizin, santonin, and amygdalin, on carbohydrate metabolism in the cotton bollworm, Helicoverpa armigera (Hübner), when diets mixed with ß-glucosides were fed to third-instar larvae. The growth of the larvae was significantly inhibited by exposure to santonin after 96 hr but not obviously affected by phlorizin and amygdalin. The midgut trehalase activities were only 51.7%, 32%, and 42.5% of that of the control after treatment with phlorizin, santonin and amygdalin at 2 mg/ml, respectively. In the hemolymph and fat body, the amount of trehalose decreased in all cases. However, the effects of santonin on the alteration of the glycogen and glucose levels as well as the activities of glycogen phosphorylase, were different than those of the other two ß-glucosides. It appears that the three ß-glucosides have different influences on the carbohydrate metabolism of cotton bollworm.

Hydrolysis of aromatic ß-glucosides by non-pathogenic bacteria confers a chemical weapon against predators.

Bacteria present in natural environments such as soil have evolved multiple strategies to escape predation. We report that natural isolates of Enterobacteriaceae that actively hydrolyze plant-derived aromatic ß-glucosides such as salicin, arbutin and esculin, are able to avoid predation by the bacteriovorous amoeba Dictyostelium discoideum and nematodes of multiple genera belonging to the family Rhabditidae. This advantage can be observed under laboratory culture conditions as well as in the soil environment. The aglycone moiety released by the hydrolysis of ß-glucosides is toxic to predators and acts via the dopaminergic receptor Dop-1 in the case of Caenorhabditis elegans. While soil isolates of nematodes belonging to the family Rhabditidae are repelled by the aglycone, laboratory strains and natural isolates of Caenorhabditis sp. are attracted to the compound, mediated by receptors that are independent of Dop-1, leading to their death. The ß-glucosides-positive (Bgl(+)) bacteria that are otherwise non-pathogenic can obtain additional nutrients from the dead predators, thereby switching their role from prey to predator. This study also offers an evolutionary explanation for the retention by bacteria of 'cryptic' or 'silent' genetic systems such as the bgl operon.

Profile and content of isoflavones on flaked and extruded soybeans and okara submitted to different drying methods.

Isoflavones (IFs) are biocompounds found in considerable amounts in soybean grains. However, to originate soybean-based materials, the grains must be subjected to numerous thermal and mechanical treatments, which can impair the IFs content. The influence of these treatments was investigated and a protocol for IFs extraction and quantification is proposed. Sequential extractions were performed on industrially pretreated soybean samples (broken, flakes, and collets), on okara submitted to different drying methods (freeze-drying, forced convection, and under vacuum), and on soybean oils extracted with hexane and ethanol. ß-glucosides levels were decreased by the thermal processes of lamination, expansion, and drying, while the aglycone forms were not affected. Lyophilization was identified as the most viable drying method for the quantification of IFs in okara. Soybean oils extracted with ethanol presented significant amounts of aglycone. Furthermore, two stages of extraction were sufficient for the recovery of the IFs from different matrices.

Human Saliva-Mediated Hydrolysis of Eugenyl-ß-D-Glucoside and Fluorescein-di-ß-D-Glucoside in In Vivo and In Vitro Models.

Eugenyl-ß-D-glucopyranoside, also referred to as Citrusin C, is a natural glucoside found among others in cloves, basil and cinnamon plants. Eugenol in a form of free aglycone is used in perfumeries, flavourings, essential oils and in medicinal products. Synthetic Citrusin C was incubated with human saliva in several in vitro models together with substrate-specific enzyme and antibiotics (clindamycin, ciprofloxacin, amoxicillin trihydrate and potassium clavulanate). Citrusin C was detected using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Citrusin C was completely degraded only when incubated with substrate-specific A. niger glucosidase E.C 3.2.1.21 (control sample) and when incubated with human saliva (tested sample). The addition of antibiotics to the above-described experimental setting, stopped Citrusin C degradation, indicating microbiologic origin of hydrolysis observed. Our results demonstrate that Citrusin C is subjected to complete degradation by salivary/oral cavity microorganisms. Extrapolation of our results allows to state that in the human oral cavity, virtually all ß-D-glucosides would follow this type of hydrolysis. Additionally, a new method was developed for an in vivo rapid test of glucosidase activity in the human mouth on the tongue using fluorescein-di-ß-D-glucoside as substrate. The results presented in this study serve as a proof of concept for the hypothesis that microbial hydrolysis path of ß-D-glucosides begins immediately in the human mouth and releases the aglycone directly into the gastrointestinal tract.