Showing papers on "Melibiose published in 1997"

The Streptomyces ATP-binding component MsiK assists in cellobiose and maltose transport.

Abstract: Streptomyces reticuli harbors an msiK gene which encodes a protein with an amino acid identify of 90% to a corresponding protein previously identified in Streptomyces lividans. Immunological studies revealed that S. lividans and S. reticuli synthesize their highest levels of MsiK during growth with cellobiose, but not with glucose. Moreover, moderate amounts of MsiK are produced by both species in the course of growth with maltose, melibiose, and xylose and by S. lividans in the presence of xylobiose and raffinose. In contrast, a recently identified cellobiose-binding protein and its distantly related homolog were only found if S. reticuli or S. lividans, respectively, was cultivated with cellobiose. Uptake of cellobiose and maltose was tested and ascertained for S. reticuli and S. lividans, but not for an msiK S. lividans mutant. However, transformants of this mutant carrying the S. reticuli or S. lividans msiK gene on a multicopy plasmid had regained the ability to transport both sugars. The data show that MsiK assists two ABC transport systems.

Simultaneous quantitation of mono-, di-and trisaccharides as their TMS ether oxime derivatives by GC-MS: I. In model solutions

Abstract: This paper describes the separation and quantitation of mono- di- and trisaccharides in model solutions. Optimized conditions for identification and determination of common sugar constituents in several natural matrices by GC-MS, from one solution by one injection, are given. Applying a 52 min temperature program to a 30 m capillary column and taking advantage of the stable ratios of the syn- and anti oximes eluting either alone or together with one or two other anomers: provides the basis of calculation for co-eluting compounds. Saccharides (5–200 ng), such as fructose, glucose, sucrose, trehalose, cellobiose, maltose, turanose, gentiobiose, palatinose, melibiose, isomaltose, erlose, raffinose, melezitose, maltotriose, panose and isomaltotriose, in the presence of each other in various ratios, have been measured as silyloxime compounds with a reproducibility of ≤5 R.S.D. %. The possibility of measuring all these saccharides as their TMS methoximes is also presented.

Proteolytic mapping and substrate protection of the Escherichia coli melibiose permease.

Abstract: The topology and substrate-induced conformational change(s) of the Na+ (Li+ or H+)-melibiose cotransporter (MelB) of Escherichia coli were investigated by limited protease digestion. To facilitate these analyses, MelB was epitope-tagged both at its carboxyl-terminus and at its amino-terminus. Limited digestion with different proteases indicates that the cytoplasmic loops connecting transmembrane domains 4-5, 6-7, and 10-11 together with the carboxyl-terminus of MelB are exposed in the cytoplasm. In contrast, periplasmic loops are highly resistant to all the proteases examined, including nonspecific proteases such as proteinase K and thermolysin. The effect of Na+ or Li+ and/or melibiose on the rate of protease digestion of the cytoplasmic loops was also analyzed. The rate of protease digestion of loop 4-5 is specifically reduced, by approximately 3-fold, by the presence of Na+ or Li+. These results suggest that loop 4-5 is near or part of the cation binding site. Moreover, the presence of both melibiose and either Na+ or Li+ further reduced the rate of protease digestion of this loop 4-5 by up to 9-fold, although no protection from protease digestion was observed when melibiose was added alone. The increase in resistance to proteases observed in the presence of the cation alone or the cation plus melibiose suggests that the interaction of the two cosubstrate with MelB results in change(s) of MelB conformation.

Purification and partial characterization of an antigen specific to Lactobacillus brevis strains with beer spoilage activity

Abstract: Certain Lactobacillus brevis strains are resistant to hop-derived compounds such as isohumulone and are able to grow in beer. In this study, we raised an antiserum against our beer spoilage laboratory strain L. brevis 578 which reacted with 23 of 24 beer spoilers and two of 13 non-spoilers in precipitation reactions using 0.5 M NaOH cell extracts. This specific antigen to the beer spoilage L. brevis strains (SABSL) was demonstrated to be located beneath the S-layer proteins by agglutination reactions using S-layer protein-stripped cells obtained by treatment with 0.1 M NaOH. SABSL was purified using an affinity column coupled with an antibody against SABSL. The purified antigen was hydrolyzed with 2 M HCl and the hydrolyzate was analyzed by thin-layer chromatography and enzymatic analysis. The results showed that SABSL contains glycerol, phosphate, glycerophosphate, d-galactose and d-glucose. d-Galactose and d-glucose accounted for 4.7% and 0.1% of the composition, respectively. Melibiose, but not mannose, inhibited the precipitation reaction. Intense precipitation reactions were obtained with fractions which did not bind to the ConA-column. These results indicate that the immunodominant component of the SABSL is galactose and the SABSL determinant is most probably a galactosylated glycerol teichoic acid. The antiserum raised against the beer spoilage strain L. brevis 578 could distinguish between Pediococcus beer spoilers and non-spoilers in precipitation reactions.

Topology of allosteric regulation of lactose permease

Abstract: Sugar transport by some permeases in Escherichia coli is allosterically regulated by the phosphorylation state of the intracellular regulatory protein, enzyme IIAglc of the phosphoenolpyruvate:sugar phosphotransferase system. A sensitive radiochemical assay for the interaction of enzyme IIAglc with membrane-associated lactose permease was used to characterize the binding reaction. The binding is stimulated by transportable substrates such as lactose, melibiose, and raffinose, but not by sugars that are not transported (maltose and sucrose). Treatment of lactose permease with N-ethylmaleimide, which blocks ligand binding and transport by alkylating Cys-148, also blocks enzyme IIAglc binding. Preincubation with the substrate analog β-d-galactopyranosyl 1-thio-β-d-galactopyranoside protects both lactose transport and enzyme IIAglc binding against inhibition by N-ethylmaleimide. A collection of lactose permease replacement mutants at Cys-148 showed, with the exception of C148V, a good correlation of relative transport activity and enzyme IIAglc binding. The nature of the interaction of enzyme IIAglc with the cytoplasmic face of lactose permease was explored. The N- and C-termini, as well as five hydrophilic loops in the permease, are exposed on the cytoplasmic surface of the membrane and it has been proposed that the central cytoplasmic loop of lactose permease is the major determinant for interaction with enzyme IIAglc. Lactose permease mutants with polyhistidine insertions in cytoplasmic loops IV/V and VI/VII and periplasmic loop VII/VIII retain transport activity and therefore substrate binding, but do not bind enzyme IIAglc, indicating that these regions of lactose permease may be involved in recognition of enzyme IIAglc. Taken together, these results suggest that interaction of lactose permease with substrate promotes a conformational change that brings several cytoplasmic loops into an arrangement optimal for interaction with the regulatory protein, enzyme IIAglc. A topological map of the proposed interaction is presented.

Extracellular melibiose and fructose are intermediates in raffinose catabolism during fermentation to ethanol by engineered enteric bacteria.

Abstract: Contrary to general concepts of bacterial saccharide metabolism, melibiose (25 to 32 g/liter) and fructose (5 to 14 g/liter) accumulated as extracellular intermediates during the catabolism of raffinose (O-alpha-D-galactopyranosyl-1, 6-alpha-D-glucopyranosyl-beta-D-fructofuranoside) (90 g/liter) by ethanologenic recombinants of Escherichia coli B, Klebsiella oxytoca M5A1, and Erwinia chrysanthemi EC16. Both hydrolysis products (melibiose and fructose) were subsequently transported and further metabolized by all three organisms. Raffinose catabolism was initiated by beta-fructosidase; melibiose was subsequently hydrolyzed to galactose and glucose by alpha-galactosidase. Glucose and fructose were completely metabolized by all three organisms, but galactose accumulated in the fermentation broth with EC16(pLOI555) and P2. MM2 (a raffinose-positive E. coli mutant) was the most effective biocatalyst for ethanol production (38 g/liter) from raffinose. All organisms rapidly fermented sucrose (90 g/liter) to ethanol (48 g/liter) at more than 90% of the theoretical yield. During sucrose catabolism, both hydrolysis products (glucose and fructose) were metabolized concurrently by EC16(pLOI555) and P2 without sugar leakage. However, fructose accumulated extracellularly (27 to 28 g/liter) at early stages of fermentation with KO11 and MM2. Sequential utilization of glucose and fructose correlated with a diauxie in base utilization (pH maintenance). The mechanism of sugar escape remains unknown but may involve downhill leakage via permease which transports precursor saccharides or novel sugar export proteins. If sugar escape occurs in nature with wild organisms, it could facilitate the development of complex bacterial communities which are based on the sequence of saccharide catabolism and the hierarchy of sugar utilization.

Replacement of isoleucine‐47 by threonine in the HPr protein of Streptococcus salivarius abrogates the preferential metabolism of glucose and fructose over lactose and melibiose but does not prevent the phosphorylation of HPr on serine‐46

Abstract: Phosphorylation of HPr on a serine residue at position 46 (Ser-46) by an ATP-dependent protein kinase has been reported in several Gram-positive bacteria, and the resulting intermediate, HPr(Ser-P), has been shown to mediate inducer exclusion in lactococci and lactobacilli and catabolite repression in Bacillus subtilis and Bacillus megaterium. We report here the phenotypic properties of an isogenic spontaneous mutant (G22.4) of Streptococcus salivarius ATCC 25975, in which a missense mutation results in the replacement of isoleucine at position 47 (Ile-47) by threonine (Thr) in HPr. This substitution did not prevent the phosphorylation of HPr on Ser-46, nor did it impede the phosphorylation of HPr on His-15 by EI or the transfer of the phosphoryl group from HPr(His-P) to other PTS proteins. However, the 147T substitution did perturb, in glucose-grown but not in galactose-grown cells, the cellular equilibrium between the various forms of HPr, resulting in an increase in the amount of free HPr at the expense of HPr(His-P)(Ser-P); the levels of HPr(His-P) and HPr(Ser-P) were not affected. Growth on melibiose was virtually identical for the wild-type and mutant strains, whereas the generation time of the mutant on the other sugars tested (glucose, fructose, mannose, lactose and galactose) increased 1.2- to 1.5-fold. The preferential metabolism of PTS sugars (glucose and fructose) over non-PTS sugars (lactose and melibiose) that is observed in wild-type cells was abolished in cells of mutant G22.4. Moreover, alpha- and beta-galactosidases were derepressed in glucose- and fructose-grown cells of the mutant. The data suggest that HPr regulates the preferential metabolism of PTS sugars over the non-PTS sugars, lactose and melibiose, through the repression of the pertinent catabolic genes. This HPr-dependent repression, however, seems to occur solely when cells are growing on a PTS sugar.

Lactose carrier mutants of Escherichia coli with changes in sugar recognition (lactose versus melibiose).

Abstract: The purpose of this research was to identify amino acid residues that mediate substrate recognition in the lactose carrier of Escherichia coli. The lactose carrier transports the alpha-galactoside sugar melibiose as well as the beta-galactoside sugar lactose. Mutants from cells containing the lac genes on an F factor were selected by the ability to grow on succinate in the presence of the toxic galactoside beta-thio-o-nitrophenylgalactoside. Mutants that grew on melibiose minimal plates but failed to grow on lactose minimal plates were picked. In sugar transport assays, mutant cells showed the striking result of having low levels of lactose downhill transport but high levels of melibiose downhill transport. Accumulation (uphill) of melibiose was completely defective in all of the mutants. Kinetic analysis of melibiose transport in the mutants showed either no change or a greater than normal apparent affinity for melibiose. PCR was used to amplify the lacY DNA of each mutant, which was then sequenced by the Sanger method. The following six mutations were found in the lacY structural genes of individual mutants: Tyr-26-->Asp, Phe-27-->Tyr, Phe-29-->Leu, Asp-240-->Val, Leu-321-->Gln, and His-322-->Tyr. We conclude from these experiments that Tyr-26, Phe-27, Phe-29 (helix 1), Asp-240 (helix 7), Leu-321, and His-322 (helix 10) either directly or indirectly mediate sugar recognition in the lactose carrier of E. coli.

A melibiose transporter and an operon containing its gene in Enterobacter cloacae.

Abstract: We detected inducible melibiose transport activity in cells of Enterobacter cloacae IID977. H+, but not Na+, was found to be the coupling cation for this transporter. We cloned and sequenced the gene encoding the melibiose transporter. A homology search of a protein sequence database revealed that this melibiose transporter has high sequence similarity with the lactose transporter (LacY) and the raffinose transporter (RafB) and has some similarity with the melibiose transporter (MelB) of Escherichia coli.

Factors regulating production of alpha-galactosidase from Bacillus sp. JF2

Abstract: Certain factors affecting the production of cell-associated alpha-galactosidase by Bacillus sp. JF2 were investigated. The intention was to maximize alpha-galactosidase activity of potential commercial application, by consecutive optimization of growth media and conditions. The highest alpha-galactosidase activity was obtained when grown on melibiose, whereas sucrose inhibited the production of alpha-galactosidase, alpha-Galactosidase production was optimally active at pH 7.5 and 55 degrees C. It was identified that a soy effluent stream could be used as the best carbon source for alpha-galactosidase by Bacillus sp. JF2.

Substrate Specificities of α-Galactosidases from Yeasts

Abstract: Twenty-nine strains of yeasts, which are capable of using galactose, melibiose, or raffinose, were screened for a-galactosidase production. Among the strains, 5 produced intracellular and extracellular α-galactosidases, and 2 produced only intracellular enzyme. Substrate specificities of these enzymes were explored using 63-α-d- galactosyl-l,4-β-d-mannotriose and 63-α-d-galactosyl-l ,4-β-d-mannotetraose. All enzymes liberated the terminal galactose from 63-α-d-galactosyl-1,4-β-D-mannotriose, but did not the stub galactose from 63-α-d-galactosyl-1,4-β-d-mannotetraose.

Different sensitivities of the sugar receptor of the lateral styloconic sensillum in fourth- and sixth-instar larvae of the spruce budworm Choristoneura fumiferana

Abstract: Female fourth- and sixth-instar larvae, Choristoneura fumiferana, were tested individually for the response of the sugar cell on the lateral styloconic sensillum to 25 mM/1 concentrations of 12 carbohydrates. The spruce budworm showed an age-related change in responsiveness of the sugar cell. The order of stimulating effectiveness for fourth-instars was melibiose > sucrose > raffinose.These storage di- and trisaccharides are present in the host plant at the beginning of budbreak. Sixth-instars responded to sucrose > fructose > m-inositol. These findings are in accordance with those of a previous behavioural study on feeding preferences of sixth-instars. The response for both melibiose and raffinose does not change from fourth- to sixth-instars; however, it does for sucrose, fructose and m- inositol.

Identification and characterization of lactic acid bacteria isolated from traditional chorizo made in Castilla-León

Abstract: A total of 516 strains of lactic acid bacteria isolated from chorizo made in Castilla-Leon (Spain) were characterized. Strains were isolated at three different stages: minced meat, half ripened chorizo and ripened chorizo. According to the Schillinger and Lucke classification, 355 strains (68.8%) were Lactobacillus sake, 85 strains (16.5%) were Lactobacillus curvatus, 32 strains (6.2%) belonged to the genus Pediococcus, and 44 strains which were not included in the previous species were grouped as Lactobacillus sp. Strains of L. sake and L. curvatus could be separated into four groups each, on the basis of fermentation of maltose and lactose. Group S1 (maltose-, lactose-negative L. sake) predominated (39.5%). Many strains of this group fermented the following carbohydrates: glucose, ribose, galactose, sucrose, melibiose and trehalose.

Raffinose-hydrolyzing activity of Aspergillus fumigatus

Abstract: Aspergillus fumigatus can utilize raffinose and melibiose as its sole carbon source. Substantial extracellular raffinolytic and invertase activities were produced during fungal growth on raffinose. These activities were induced also by sucrose, melibiose and wheat straw but not by glucose. Hydrolysis of both raffinose and sucrose was optimal at pH 4.5 to 5.5 and 55 to 60°C. Melibiose was also hydrolyzed by the culture filtrate. The crude enzyme retained about 80% of the original activity (2 U/mL) after incubation for 2 h at 50°C.

Homogeneous catalyst for DNA hydrolysis (4). Efficient DNA hydrolysis by lanthanide--saccharide complexes.

Abstract: Homogeneous and neutral solutions are prepared by mixing Ce(NH4)2(NO3)6 with either isomaltose, melibiose, gentiobiose, palatinose, mannitol, sorbitol, galactitol, or glucamine in pH 7 hepes buffer ([Ce(IV)]0/[monomeric residue of saccharide]0 = 1). In contrast, amylose, cyclodextrins, maltose, glucose, and fructose provide only heterogeneous mixtures. The homogeneous solution of 1:1 Ce(IV)/glucamine system is active for DNA hydrolysis: the pseudo-first-order rate constant for the hydrolysis of thymidylyl(3'-->5')thymidine at pH 7.0 and 50 degrees C is 0.010 h-1, when [Ce(IV)]0 = [glucamine]0 = 10 mmol dm-3. The DNA-hydrolyzing activity decreases in the following order: glucamine > isomaltose, melibiose, gentiobiose >> palatinose, mannitol, sorbitol, galactitol.

Chromosomal deletions in Streptococcus mutans.

Abstract: The oral bacterium Streptococcus mutans possesses the ability to ferment a wide range of carbohydrates, which results in the production of acids that can cause demineralisation of tooth enamel and subsequent dental caries. However, it has been shown that approximately 11% of independent isolates of S. mutans from around the world lack the ability to ferment melibiose, raffinose, and on occasion β-glucosides (1,4). This is due to a deletion which extends through a region of the genome that includes the multiple sugar metabolism (msm) operon involved in the uptake and metabolism of a number of sugars (3). The msm region has been reintroduced to the deletion mutants by transformation and the ability to grow on galactose and β-glucosides was also restored, showing the linkage of the respective genes. The extent of the deletion is further being investigated with the use of pulsed field gel electrophoresis and Southern hybridizations with known S. mutans genes as probes.