Influence of various bile salts on ββ-glucuronidase activity of intestinal bacteria

While the decrease of the β-glucuronidase activity of sonicated cells of Clostridium perfringens and Escherichia coli was obvious for sodium deoxycholate (DC), it was not so obvious for other bile salts (sodium glycocholate and sodium cholate). The enzyme activity of intact cells of these bacteria was significantly enhanced by the presence of DC, but not by the other bile salts in the buffer. These results suggest that the permeability of the bacterial cells is increased more by the presence of DC than by other bile salts.     

Fluorogenic assay for immediate presumptive detection of Escherichia coli in urine

A rapid and direct fluorogenic assay was used to detect Escherichia coli in urine. Most clinical isolates of E. coli produce β-glucuronidase, whereas almost all other enterobacteria lack the enzyme. Spectrofluorimetric assay of β-glucuronidase, without previous induction, was performed on growing and starved uropathogenic E. coli in artificial urine. The presence of 10³ cfu ml^-1 of E. coli in urine was detected by β-glucuronidase activity in less than 1 h. These results indicate that β-glucuronidase is a rapid, specific and sensitive indicator of the presence of E. coli in urine, and provide additional information on the biological state of the infecting bacterial population.     

The influence of incubation pH on the activity of rat and human gut flora enzymes

The activities of three bacterial biotransformation enzymes (β-glucuronidase, β-glucosidase, nitrate reductase) were determined in suspensions of rat caecal contents or human faeces over the pH range 6–8. All three enzymes were influenced by pH, as exemplified by β-glucosidase activity which diminished as pH increased. In other instances the rat and human flora showed distinct profiles, with nitrate reductase activity undetectable in human faeces below pH 6–6, whereas the rat caecal flora displayed optimal reduction of nitrate around neutrality. The most pronounced host-species difference was found with β-glucuronidase, which showed maximal activity at pH 6–0 in human faecal bacteria, while the rat caecal flora expressed greatest activity at pH 8–0. All three enzyme activities were associated with that fraction of rat caecal or human faecal material sedimented by centrifugation at 5000 g for 15 min, with little or no metabolism occurring in the 11000 g supernatant fluid. The results demonstrate that pH has a pronounced effect on the enzymic activity of bacterial preparations from rat and human sources.     

Developmental changes to gut microflora metabolism in mice

W.E. BRENNAN-CRADDOCK, A.K. MALLETT, I.R. ROWLAND AND S. NEALE. 1992. Developmental changes in the activities of bacterial nitrate reductase, nitroreductase and β-glucuronidase and their response to fermentable dietary fibre, were investigated in caecal contents from suckling mice (2-week-old) and in mice aged 4–24 weeks fed either a purified fibre-free diet or that diet supplemented with 5% (w/w) pectin. There was no apparent age-related trend common to the three enzymes studied. Nitrate reductase activity in the mice fed the fibre-free diet did not markedly alter with age. Pectin administration, however, was associated with a significant increase in nitrate reductase activity, particularly in 4-week-old mice. Nitroreductase activity exhibited an overall upward trend in mice from 2 to 12 weeks and thereafter decreased. Caecal β-glucuronidase activity in mice increased sharply between 2 weeks and 4 weeks of age, thereafter not changing significantly until the 24th week. Pectin feeding had no consistent effect on activities either of nitroreductase or β-glucuronidase. The changes in enzyme activities with age were not related to the concentration of bacteria in the caecum, which was highest in the 2-week-old mice.
We conclude that the weaning is a period in which marked changes in caecal bacterial enzyme activities can occur.     

A note on lactobacilli and β-glucuronidase activity in the intestinal contents of mice

A comparison was made of caecal β-glucuronidase activity in the caecal contents of mice with or without lactobacilli as members of the normal microflora. Male mice that did not have lactobacilli as intestinal inhabitants had approximately 52% more caecal β-glucuronidase activity than their female counterparts. Colonization of male mice by lactobacilli reduced the β-glucuronidase activity to that of female mice.     

Effect of two levels of transgalactosylated oligosaccharide intake in rats associated with human faecal microflora on bacterial glycolytic activity, end-products of fermentation and bacterial steroid transformation

The effects of two levels of transgalactosylated oligosaccharide (TOS) intake on bacterial glycolytic activity, end products of fermentation and bacterial steroid transformation were studied in rats associated with a human faecal flora. Rats were fed a human-type diet containing 0, 5 or 10% TOS. Caecal pH decrease correlated with the amount of TOS in the diet. Intake of the TOS diet induced a decrease in blood cholesterol and a strong increase in β-galactosidase activity in the hindgut. TOS fermentation led to production of hydrogen and short chain fatty acids, whereas ammonia and branched-chain fatty acids were decreased. A diet containing 10% TOS increased caecal lactic acid concentrations and reduced β-glucuronidase activities and steroid transformation.     

Glycosaminoglycan Storage in Cultured Neonatal Murine Mucopolysaccharidosis Type VII Neuroglial Cells and Correction by β-Glucuronidase Gene Transfer

Abstract: The inherited deficiency of β-glucuronidase activity causes the lysosomal storage disorder mucopolysaccharidosis (MPS) type VII (Sly disease). The sequential catabolism of glycosaminoglycans in lysosomes is blocked, and undegraded substrates accumulate in cells of many tissues, including neurons and glia in the brain. To evaluate the deficient metabolic pathway, primary cultures of mixed brain cells were established from newborn MPS VII mice. β-Glucuronidase levels and glycosaminoglycan accumulation were studied in normal, carrier, and MPS VII cells. Retroviral vector-mediated transfer of a normal β-glucuronidase cDNA corrected the enzymatic deficiency in MPS VII cells and restored glycosaminoglycan catabolism to normal. High levels of β-glucuronidase expression were sustained in vector-corrected nondividing glial cell cultures for >2 months. These studies provide an in vitro model for evaluating somatic gene transfer in neural cells affected in mucopolysaccharidoses.     

Plant endogenous β-glucuronidase activity: how to avoid interference with the use of theE. coli β-glucuronidase as a reporter gene in transgenic plants

We have detected a plant β-glucuronidase activity, present in several tissues and organs of plant species belonging to different families. The fluorimetric β-glucuronidase assay was used to partially characterize this activity in post-ribosomal supernatants of tobacco leaves. The tobacco activity is very stable at low temperatures, but quickly inactivated above 45°C. It is relatively resistant to proteases and insensitive to-SH group reagents and to ionic conditions. It does not require, nor is it inhibited by, divalent cations. Although these properties are shared by theEscherichia coli β-glucuronidase, the two activities can be distinguished by: (i) their different sensitivity to the specific inhibitor saccharic acid-1,4-lactone; (ii) their different thermal stability (iii) their different pH optima (5.0 for the plant activity and close to neutral for the bacterial enzyme). Therefore, under appropriate experimental conditions, it should be possible to assay theE. coli β-glucuronidase in transgenic plants without interference from the endogenous plant activity.     

Co-transformation of Metarhizium anisopliae by electroporation or using the gene gun to produce stable GUS transformants

Abstract The potential of β-glucuronidase as a molecular marker for studying the environmental microbiology of entomopathogenic fungi was assessed. Metarhizium anisopliae was stably co-transformed with plasmids (pNOM102 and pBENA3) containing the β-glucuronidase and benomyl resistance (β-tubulin) genes, using both electroporation and biolistic delivery systems, and it was confirmed that the expressed phenotypes were not exhibited by ten randomly chosen indigenous North-American isolates. In spite of random and multiple integrations, the co-transformants showed normal growth rates and retained their pathogenicity to insects. β-Glucuronidase activity in the co-transformants was used to detect histochemically the presence of fungal hyphae in infected host insects ( Bombyx mori ) and thus provides a practical means of marking genetically engineered pathogens for field trials.     

Optimum conditions for the release and regeneration of protoplasts of Alternaria alternata

Best release of Alternaria alternata protoplasts was obtained when 20 h old mycelia were incubated in a hydrolytic enzyme mixture of Novozym 234, Driselase, and β -glucuronidase. Numbers of nuclei/protoplast varied but generally decreased with increased time of incubation. While salts, were better osmotic stabilizers for protoplast release, sucrose and sugar alcohols were better for regeneration.     

胆汁β-葡萄糖醛酸苷酶的性质及其计算荧光法

 胆汁经Sephadex G-25分子筛层析和DEAE-纤维素吸附处理,除去其中直接胆红素及胆汁酸盐,然后对其中的β-葡萄糖醛酸苷酶(GUSB)的性质进行了详细研究:该酶的最适温度为56℃;最适pH为4.5;以4mu Gr为底物测得Km为0.68mmol/L;直接胆红素是其竞争性抑制剂,其k_i=2.04×10~(-3)mmol/L;PI_1=6.0—6.2,PI_2=7.2—7.4,MW=280kD。根据竞争性抑制的米氏方程式设计建立了一个在直接胆红素并存的条件下,定量测定胆汁GUSB的计算荧光法,准确性为97.97±1.79％,重复性CV=1.2％,测定时间大大缩短。利用本法在pH4.5和7.0条件下测定了20例胆红素胆结石和17例胆固醇胆结石患者的胆汁GUSB的真实活性。结果指出:不论在pH4.5或7.0条件下,前组活性均显著高于后组(P<0.01)。证明胆汁中高GUSB活性与胆红素胆结石的形成有密切关系。     

Silicon carbide fiber-mediated DNA delivery into plant cells

Summary Silicon carbide fiber-mediated delivery of DNA into intact plant cells was investigated. Black Mexican Sweet (BMS) maize (Zea mays) and tobacco (Nicotiana tabacum) suspension culture cells were vortexed in the presence of liquid medium, plasmid DNA encoding -glucuronidase (GUS), and silicon carbide fibers. Penetration of BMS cells by the silicon carbide fibers was observed by scanning electron microscopy of vortexed cells. Following fiber and DNA treatment, BMS cells transiently expressed GUS activity at a mean frequency of 139.5 units (one unit = one blue cell or one colony of blue cells) per sample. Treated tobacco cells expressed an average of 373 GUS units per sample. Untreated controls did not exhibit GUS activity. These results indicate that the silicon carbide fibers-vortex procedure can be used to rapidly and inexpensively deliver foreign DNA into intact plant cells for investigations of transient gene expression.Abbreviations BMS Black Mexican Sweet maize suspension cultures - MS Murashige and Skoog salts - GUS -glucuronidase - 2,4-D 2,4 dichlorophenoxyacetic acid     

The effect of bile salts on survival and morphology of a potential probiotic strain Lactobacillus acidophilus M92

Bile tolerance is an important criterion in the selection of microbial strains for probiotic use. The survival and morphological changes of a potential probiotic strain, Lactobacillus acidophilus M92, in the presence of bile salts were examined. Lactobacillus acidophilus M92 has shown a satisfactory degree of tolerance against oxgall and individual bile salts tested, especially to taurocholate. The higher resistance of L. acidophilus M92 against taurine-conjugated bile salts relative to deconjugated and glycine-conjugated bile salts was attributed to its reaction to the stronger acidity of the former. Furthermore, bile salt hydrolase (BSH) was active when L. acidophilus M92 was grown in the presence of sodium taurocholate. The rate of BSH activity was highest at the exponential growth phase. It was hypothesised that BSH activity may be important for the bile salt resistance of this strain. The colonial and cellular morphology may also be a valuable parameter in the selection of bile salt-resistant Lactobacillus strains for probiotic use. Smooth (S) and rough (R) colonies, appeared in the original L. acidophilus M92 bacterial culture and demonstrated a different degree of bile tolerance. Rough colonies were more sensitive to bile salts than smooth ones. The R colony cells assumed a round form, probably induced by gaps in the cell wall caused by the cytotoxicity of glycodeoxycholate. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characterization of inhibitor(s) of β-glucuronidase enzyme activity in <Emphasis Type="Italic">GUS</Emphasis>-transgenic wheat

The uidA gene, encoding for β-glucuronidase (GUS), is the most frequently used reporter gene in plants. As a reporter enzyme, GUS can be assayed both qualitatively and quantitatively. In wheat, there are numerous reports of failure in detecting GUS enzyme activity in tissues of transgenic plants, while other reports have suggested presence of β-glucuronidase inhibitor(s) in wheat tissues. In the present study, we show that the β-glucuronidase enzyme activity is not only tissue-specific but also genotype-dependent. Our data demonstrate that the glucuronic acid could be the candidate inhibitor for β-glucuronidase enzyme activity in wheat leaves and roots. It should be noted that the assays to detect β-glucuronidase enzyme activity in wheat should be interpreted carefully. Based on the data of our present study, we recommend studying the chemical pathways, the unintended effects and the possible loss-of-function of any candidate transgene prior to transformation experiments.     

Specific detection of Escherichia coli and Shigella species using fragments of genes coding for β-glucuronidase

Abstract The occurrence of β-glucuronidase activity, a main characteristic of Escherichia coli and the presence of the uid chromosomal region of E. coli , coding for this enzyme, were tested on representative members of enteric bacteria. DNA hybridization techniques using uid probes and ampplification experiments of uidA gene by the polymerase chain reaction (PCR) confirmed the specificity of uid genes fro E. coli and Shigella spp. (i.e., S. boydii, S. dysenteriae, S. flexneri and S. sonnei ), independent of the β-glucuronidase phenotype of bacterial strains. This specificity seemed to be conserved when studies were extended to a wide range of bacteria. It was not possible to distinguish E. coli from Shigella spp. The detection sensitivity using double stranded DNA radiolabeled probes was 3 × 10⁴ bacteria and could be brought down to 8 bacteria by PCR. Thus, the uid genes appeared to be ideal candidates for DNA probes technology to detect E. coli-Shigella species.     

Histochemical investigation of β-glucuronidase in culture cells and regenerated plants of Scutellaria baicalensis Georgi

 Strong activity of β-glucuronidase first appeared in the epidermal and glandular hair cells of leaf primordia regenerated from callus of Scutellaria baicalensis Georgi. Leaf primordia matured rapidly in culture to form shoots within 1 month in which both the mesophyll cells and the glandular hairs were deeply stained. Leaves predominantly accumulated β-glucuronidase in both glandular hair cells and mesophyll cells. β-Glucronidase activity in leaves was higher in the summer and decreased in the winter. The stem section collected in the summer had a different β-glucuronidase distribution pattern from that of the root in that in the former strong activity appeared in the periderm cells and collenchyma cells which was decreasingly dispersed into the phloem layer cells. In the winter, β-glucronidase activity decreased compared to that in summer. It can be argued that the distribution of β-glucuronidase in this plant is closely linked with the defense against pathogens: it is a starting key enzyme which may act together with the flavonoids, which play an important role as a proton donor for the detoxification metabolism of H₂O₂. Received: 1 December 1998 / Revision received: 10 March 1999 / Accepted: 14 June 1999     

Characterization of the new β-glucuronidase from <Emphasis Type="Italic">Streptococcus equi</Emphasis> subsp. <Emphasis Type="Italic">zooepidemicus</Emphasis>

Recently, a new gene encoding β-glucuronidase from Streptococcus equi subsp. zooepidemicus (SEZ) was identified and expressed in Escherichia coli. In this paper, the characterization of the enzyme is described. Specific enzyme activity was 120,000 U/mg purified protein at 37°C and pH = 7.0. The temperature and pH value, at which the enzyme has the highest specific activity, were determined and were found to be approximately 52°C and 5.6, respectively. The mutant strain SEZ glcHis was designed for the efficient isolation of β-glucuronidase from S. equi subsp. zooepidemicus. It was observed that the specific activity of β-glucuronidase in the cytoplasmic extract of a mutated strain was about 45% lower than in the cytoplasmic extract of a wild-type strain. The specific activity of purified β-glucuronidase from SEZ glcHis was four times as low as β-glucuronidase purified from E. coli. Comparing the specific activity of purified streptococcal β-glucuronidase from E. coli with E. coli β-glucuronidase (the enzyme with the highest specific activity was supplied by Sigma), the former is 1.8 higher than the latter.     

The assumed assimilation of cholesterol by Lactobacilli and Bifidobacterium bifidum is due to their bile salt-deconjugating activity.

To study the mechanism of the propsed assimilation of cholesterol, we cultured various strains of Lactobacillus acidophilus and a Bifidobacterium sp. in the presence of cholesterol and oxgall. During culturing, both cholesterol and bile salts were precipitated. Because of bacterial bile salt deconjugation, no conjugated bile salts were observed in either the culture fluids or the pellets. During incubation, the cell count and optical density decreased. The degree of precipitation of bile salts and of cholesterol was dependent on the culture conditions. If L. acidophilus RP32 was cultured under acidifying conditions, the degree of precipitation of deconjugated bile salts was higher than if the pH was maintained at 6.0. Under acidifying conditions, cholesterol was coprecipitated with the bile salts, whereas in pH-controlled cultures, no coprecipitation of cholesterol was observed. From control experiments with different mixtures of bile salts, it appeared that coprecipitation of cholesterol during culturing was a result of formation of deconjugated bile salts, which have a decreased solubility at pH values lower than 6.0. It is concluded that the removal of cholesterol from the culture medium by L. acidophilus RP32 and other species is not due to bacterial uptake of cholesterol, but results from bacterial bile salt-deconjugating activity.