Growth of Azospirillum irakense KBC1 on the aryl beta-glucoside salicin requires either salA or salB. (1/54)

The rhizosphere nitrogen-fixing bacterium Azospirillum irakense KBC1 is able to grow on pectin and beta-glucosides such as cellobiose, arbutin, and salicin. Two adjacent genes, salA and salB, conferring beta-glucosidase activity to Escherichia coli, have been identified in a cosmid library of A. irakense DNA. The SalA and SalB enzymes preferentially hydrolyzed aryl beta-glucosides. A Delta(salA-salB) A. irakense mutant was not able to grow on salicin but could still utilize arbutin, cellobiose, and glucose for growth. This mutant could be complemented by either salA or salB, suggesting functional redundancy of these genes in salicin utilization. In contrast to this functional homology, the SalA and SalB proteins, members of family 3 of the glycosyl hydrolases, show a low degree of amino acid similarity. Unlike SalA, the SalB protein exhibits an atypical truncated C-terminal region. We propose that SalA and SalB are representatives of the AB and AB' subfamilies, respectively, in glycosyl hydrolase family 3. This is the first genetic implication of this beta-glucosidase family in the utilization of beta-glucosides for microbial growth.  (+info)

Lipid composition determines the effects of arbutin on the stability of membranes. (2/54)

Arbutin (hydroquinone-beta-D-glucopyranoside) is an abundant solute in the leaves of many freezing- or desiccation-tolerant plants. Its physiological role in plants, however, is not known. Here we show that arbutin protects isolated spinach (Spinacia oleracea L.) thylakoid membranes from freeze-thaw damage. During freezing of liposomes, the presence of only 20 mM arbutin led to complete leakage of a soluble marker from egg PC (EPC) liposomes. When the nonbilayer-forming chloroplast lipid monogalactosyldiacylglycerol (MGDG) was included in the membranes, this leakage was prevented. Inclusion of more than 15% MGDG into the membranes led to a strong destabilization of liposomes during freezing. Under these conditions arbutin became a cryoprotectant, as only 5 mM arbutin reduced leakage from 75% to 20%. The nonbilayer lipid egg phosphatidylethanolamine (EPE) had an effect similar to that of MGDG, but was much less effective, even at concentrations up to 80% in EPC membranes. Arbutin-induced leakage during freezing was accompanied by massive bilayer fusion in EPC and EPC/EPE membranes. Twenty percent MGDG in EPC bilayers completely inhibited the fusogenic effect of arbutin. The membrane surface probes merocyanine 540 and 2-(6-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl)amino)hexanoyl-1-hexadecanoyl-sn-glycero-3-phosph ocholi ne (NBD-C(6)-HPC) revealed that arbutin reduced the ability of both probes to partition into the membranes. Steady-state anisotropy measurements with probes that localize at different positions in the membranes showed that headgroup mobility was increased in the presence of arbutin, whereas the mobility of the fatty acyl chains close to the glycerol backbone was reduced. This reduction, however, was not seen in membranes containing 20% MGDG. The effect of arbutin on lipid order was limited to the interfacial region of the membranes and was not evident in the hydrophobic core region. From these data we were able to derive a physical model of the perturbing or nonperturbing interactions of arbutin with lipid bilayers.  (+info)

Expression of the phospho-beta-glycosidase ArbZ from Lactobacillus delbrueckii subsp. lactis in Lactobacillus helveticus: substrate induction and catabolite repression. (3/54)

ArbZ from Lactobacillus delbrueckii subsp. lactis was previously shown to enable utilization of the beta-glucoside arbutin by Escherichia coli. The arbZ gene was cloned and expressed in the industrially used beta-glucoside-negative strain Lactobacillus helveticus 3036(62). The transformants were able to ferment not only arbutin, but also cellobiose, salicin and methyl-beta-glucoside (MbetaGlc). Cleavage of beta-glucosides by the transformants depended on the integrity of the cytoplasmic membrane, whereas in cell-free extracts only C(6)-phosphorylated substrates were hydrolysed. This suggested that ArbZ is a phospho-beta-glycosidase. ArbZ activity in transformants of Lb. helveticus was subject to substrate induction mediated by the beta-glucosides arbutin, salicin and MbetaGlc, whereas cellobiose or the beta-galactoside lactose had no inducing effect. Northern blot analysis proved that induction by MbetaGlc was due to enhanced transcription of arbZ. Catabolite repression of arbZ induction was observed with glucose, mannose, fructose and galactose. The induction kinetics observed in the presence of these sugars indicated that at least two different mechanisms are operative in catabolite repression of arbZ in Lb. helveticus.  (+info)

Analysis of the beta-glucoside utilization (bgl) genes of Shigella sonnei: evolutionary implications for their maintenance in a cryptic state. (4/54)

The pattern of expression of the genes involved in the utilization of aryl beta-glucosides such as arbutin and salicin is different in the genus Shigella compared to Escherichia coli. The results presented here indicate that the homologue of the cryptic bgl operon of E. coli is conserved in Shigella sonnei and is the primary system involved in beta-glucoside utilization in the organism. The organization of the bgl genes in S. sonnei is similar to that of E. coli; however there are three major differences in terms of their pattern of expression. (i) The bglB gene, encoding phospho-beta-glucosidase B, is insertionally inactivated in S. sonnei. As a result, mutational activation of the silent bgl promoter confers an Arbutin-positive (Arb(+)) phenotype to the cells in a single step; however, acquiring a Salicin-positive (Sal(+)) phenotype requires the reversion or suppression of the bglB mutation in addition. (ii) Unlike in E. coli, a majority of the activating mutations (conferring the Arb(+) phenotype) map within the unlinked hns locus, whereas activation of the E. coli bgl operon under the same conditions is predominantly due to insertions within the bglR locus. (iii) Although the bgl promoter is silent in the wild-type strain of S. sonnei (as in the case of E. coli), transcriptional and functional analyses indicated a higher basal level of transcription of the downstream genes. This was correlated with a 1 bp deletion within the putative Rho-independent terminator present in the leader sequence preceding the homologue of the bglG gene. The possible evolutionary implications of these differences for the maintenance of the genes in the cryptic state are discussed.  (+info)

Effect of microorganisms isolated from the upper gut of malnourished children on intestinal sugar absorption in vivo. (5/54)

The effect of microorganisms isolated from the upper gastrointestinal tract of malnourished children on intestinal sugar absorption was studied in rats in vivo. Pure cultures of organisms were grown overnight in a nutrient broth and the resultant supernatant fluid which contained microorganisms in similar numbers to those found in the patients was used as the basic solution for jejunal perfusions which were done in anesthetized adult Wistar rats. The substrate used was arbutin (p-hydroxphenyl-beta-glucoside), a recognized marker of intestinal active sugar transport. Of the gram-positive cocci studied, only the saprophyte, Staphylococcus saprophyticus, did not adversely affect the intestinal absorption of arbutin. The only gram-positive rod studied, a lactobacillus, also significantly inhibited arbutin absorption. Of the Enterobacteriaciae studied, Salmonella paratyphi B, a Shigella and Proteus sp. did not affect arbutin absorption. All the species of Escherichia coli studied, including a nonpathogenic variety, inhibited absorption. Klebsiella sp. and Pseudomonas sp. were also effective. Of the Candida sp., C. albicans and C. parapsilosis were inhibitory while C. tropicalis was not. These results suggest that microorganisms not generally considered enteropathogenic may adversely affect intestinal function when present in the lumen of the gut in excessive numbers and contribute to the production of diarrhea in children with malnutrition.  (+info)

Phenotypic and genotypic properties of the genus Hafnia. (6/54)

The present study characterised 73 Hafnia alvei isolates and five Escherichia isolates (originally identified as H. alvei) isolated from cases of diarrhoeal disease by the International Centre for Diarrhoeal Disease Research Branch (ICDDRB) in Bangladesh. Based upon the hydrolysis of arbutin and aesculin and the fermentation of salicin and D-arabinose, four distinct biotypes could be recognised among the 73 H. alvei isolates tested; biotype 1 (D-(-)-arabinose-positive only) accounted for 75% of all isolates analysed. Hydrolysis of aglycone compounds such as arbutin, salicin and aesculin appeared to be associated with expression of beta-glucosidase activity. ICDDRB isolates, when compared with type or reference strains of H. alvei, were shown not to belong to the genus Hafnia based upon resistance to Hafnia-specific bacteriophage 1672, possession of the phoE gene, expression of glutamate decarboxylase activity and significant 16S rDNA sequence divergence (approximately 8%) from the type strain, ATCC 13337T. True H. alvei strains, implicated in outbreaks of diarrhoeal disease in Canada, lacked the eaeA gene in contrast to ICDDRB isolates. Twenty-two H. alvei isolates were selected for further study. Based upon partial 16S rDNA sequencing, these 22 isolates fell into two genomic groups (genomospecies), identical to DNA groups previously established by DNA hybridisation studies. Markers such as motility, biotype, or enzymic or carbohydrate fermentation patterns did not correlate totally with DNA grouping, although malonate utilisation appeared to be the single best discriminatory phenotype. The results indicate that the genus Hafnia is heterogeneous and there do not appear to be any laboratory data available specifically linking these organisms to gastro-enteritis.  (+info)

Probing suggested catalytic domains of glycosyltransferases by site-directed mutagenesis. (7/54)

The plant enzyme arbutin synthase isolated from cell suspension cultures of Rauvolfia serpentina and heterologously expressed in Escherichia coli is a member of the NRD1beta family of glycosyltransferases. This enzyme was used to prove, by site-directed mutagenesis, suggested catalytic domains and reaction mechanisms proposed for enzyme-catalyzed glycosylation. Replacement of amino acids far from the NRD domain do not significantly affect arbutin synthase activity. Exchange of amino acids at the NRD site leads to a decrease of enzymatic activity, e.g. substitution of Glu368 by Asp. Glu368, which is a conserved amino acid in glycosyltransferases located at position 2 and is important for enzyme activity, does not serve as the nucleophile in the catalytic centre as proposed. When it is replaced by Ala, the resulting mutant enzyme E368A exhibits comparable activity as found for E368D in respect to vanillin. Enzyme activities of wild-type and E368A towards several substrates were not affected at the same level. His360 at position 1 of NRD1beta glycosyltransferases occupies a more crucial role as expected. When it is exchanged against other basic amino acids such as Lys or Arg the enzyme activity decreases approximately 1000-fold. Replacement of His360 by Glu leads to a mutant enzyme (H360E) with an approximately 4000-fold lower activity compared with the wild-type. This mutein still produces a beta-glucoside, not an alpha-glucoside and therefore indicates that generation of the typical E-E motif of NRD1alpha glycosyltransferases does not convert a NRD1beta enzyme into a NRD1alpha enzyme. The presented data do not support several suggestions made in the literature about catalytic amino acids involved in the glycosyltransfer reaction.  (+info)

Syntheses of arbutin-alpha-glycosides and a comparison of their inhibitory effects with those of alpha-arbutin and arbutin on human tyrosinase. (8/54)

The effects of 4-hydroxyphenyl alpha-glucopyranoside (alpha-arbutin) and 4-hydroxyphenyl beta-glucopyranoside (arbutin) on the activity of tyrosinase from human malignant melanoma cells were examined. The inhibitory effect of alpha-arbutin on human tyrosinase was stronger than that of arbutin. The K(i) value for alpha-arbutin was calculated to be 1/20 that for arbutin. We then synthesized arbutin-alpha-glycosides by the transglycosylation reaction of cyclomaltodextrin glucanotransferase using arbutin and starch, respectively, as acceptor and donor molecules. The structural analyses using 13C- and 1H-NMR proved that the transglycosylated products were 4-hydroxyphenyl beta-maltoside (beta-Ab-alpha-G1) and 4-hydroxyphenyl beta-maltotrioside (beta-Ab-alpha-G2). These arbutin-alpha-glycosides exhibited competitive type inhibition on human tyrosinase, and their K(i) values were calculated to be 0.7 mM and 0.9 mM, respectively. These arbutin-alpha-glycosides possessed stronger inhibitory activity than arbutin, but less activity than alpha-arbutin. These results suggested that the alpha-glucosidic linkage of hydroquinone-glycosides plays an important role in the inhibitory effect on human tyrosinase.  (+info)

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