Utilization Of Raffinose Research Articles

Different utilization of glucose and raffinose inBifidobacterium breve andBifidobacterium animalis

Growth of 2 strains of Bifidobacterium breve and 4 strains of Bifidobacterium animalis was evaluated with glucose, raffinose or a mixture of glucose and raffinose as substrates. All strains of B. animalis and one strain of B. breve grew more slowly on glucose than on raffinose or the mixture of glucose and raffinose; one strain of B. breve grew more rapidly on the mixture of glucose and raffinose than on raffinose while 2 strains of B. animalis grew faster on raffinose than on the mixture of raffinose and glucose. Both strains of B. breve utilized glucose and raffinose simultaneously. In contrast, all strains of B. animalis strains displayed atypical growth with rapid utilization of raffinose followed by slow utilization of glucose. The cell morphology of all strains of B. animalis was affected by the sugar used for cultivation: tiny and rather spherical cells were observed on glucose while on media with raffinose the cells were large and had the species-specific shape. Description of preferential utilization of various saccharides by bifidobacteria can contribute to the development of new synbiotic preparations and new cultivation media for bifidobacteria.

Molecular characterization, technological properties and safety aspects of enterococci from 'Hussuwa', an African fermented sorghum product

To identify enterococci from Hussuwa, a Sudanese fermented sorghum product, and determine their technological properties and safety for possible inclusion in a starter culture preparation. Twenty-two Enterococcus isolates from Hussuwa were identified as Enterococcus faecium by using phenotypic and genotypic tests such as 16S rDNA gene sequencing, RAPD-PCR and restriction fragment length polymorphism of the 16S/23S intergenic spacer region fingerprinting. Genotyping revealed that strains were not clonally related and exhibited a considerable degree of genomic diversity. Some strains possessed useful technological properties such as production of bacteriocins and H2O2 or utilization of raffinose and stachyose. None produced alpha-amylase or tannase. A safety investigation revealed that all strains were susceptible to the antibiotics ampicillin, gentamicin, chloramphenicol, tetracycline and streptomycin, but some were resistant to ciprofloxacin, erythromycin, penicillin and vancomycin. Production of biogenic amines or presence of genes encoding virulence determinants occurred in some strains. Enterococcus faecium strains are associated with fermentation of Sudanese Hussuwa. Some strains exhibited useful technological properties such as production of antimicrobial agents and fermentation of indigestible sugars, which may aid in stabilizing and improving the digestibility of the product respectively. Enterococci were shown to play a role in the fermentation of African foods. While beneficial properties of these bacteria are indicated, their presence in this food may also imply a hygienic risk as a result of antimicrobial resistances or presence of virulence determinants.

MIG1-dependent and MIG1-independent regulation of GAL gene expression in Saccharomyces cerevisiae: role of Imp2p.

Imp2p (Yil154c) is a transcriptional activator involved in glucose derepression of the maltose, galactose and raffinose utilization pathways and in resistance to thermal, oxidative or osmotic stress. We analysed the role of Imp2 in the regulation of GAL genes. Imp2 was shown to have a positive effect on glucose derepression of Leloir pathway genes and their activator gene GAL4. The effect of Imp2 on galactose metabolism was shown to be partially dependent on Mig1p. The Mig1-independent role depends on Nrg1p. However, disruption of both MIG1 and NRG1 only partially relieves the glucose repression of GAL genes in the Deltaimp2 mutant, indicating that Imp2 must also have other function(s). Moreover, the interaction between IMP2 and GAL6/BLH1, a recently isolated gene involved in the regulation of GAL genes that shares with Imp2 the ability to protect cells from the glycopeptide bleomycin, was also analysed. The results suggest a major role of Imp2 in a GAL6-independent pathway.

Utilization of raffinose and melibiose by a mutant ofSaccharomyces cerevisiae

The mutant Saccharomyces cerevisiae ATCC 36858 was used in the production of ethanol and/or fructose from synthetic media in batch processes with raffinose, melibiose or sucrose. The mutant was able to hydrolyze all the sugars used and to selectively ferment glucose and galactose to ethanol while fructose accumulated in the fermentation medium. The fructose yield was above 89% of the theoretical value in the media with either raffinose or sucrose, when initial concentrations were between 131.5 g dm−3 and 242.0 g dm−3. The ethanol yields were 82% and 77% of the theoretical values in the media with melibiose and sucrose, respectively, and about 72% of the theoretical value when raffinose was used. The fructose fraction in the carbohydrate content of the produced syrups was more than 96% when raffinose concentration was below 189.1 g dm−3. However, even at a sucrose concentration of 187.9 g dm−3, the produced syrup contained 100% fructose. Some oligosaccharides were also produced in all tested media. The produced oligosaccharides were consumed by the end of the fermentation process. These findings can be useful in the production of ethanol and high fructose syrups using raw materials based on sucrose and raffinose such as molasses. Copyright © 2003 Society of Chemical Industry

Control of inducer accumulation plays a key role in succinate-mediated catabolite repression in Sinorhizobium meliloti.

The symbiotic, nitrogen-fixing bacterium Sinorhizobium meliloti favors succinate and related dicarboxylic acids as carbon sources. As a preferred carbon source, succinate can exert catabolite repression upon genes needed for the utilization of many secondary carbon sources, including the alpha-galactosides raffinose and stachyose. We isolated lacR mutants in a genetic screen designed to find S. meliloti mutants that had abnormal succinate-mediated catabolite repression of the melA-agp genes, which are required for the utilization of raffinose and other alpha-galactosides. The loss of catabolite repression in lacR mutants was seen in cells grown in minimal medium containing succinate and raffinose and grown in succinate and lactose. For succinate and lactose, the loss of catabolite repression could be attributed to the constitutive expression of beta-galactoside utilization genes in lacR mutants. However, the inactivation of lacR did not cause the constitutive expression of alpha-galactoside utilization genes but caused the aberrant expression of these genes only when succinate was present. To explain the loss of diauxie in succinate and raffinose, we propose a model in which lacR mutants overproduce beta-galactoside transporters, thereby overwhelming the inducer exclusion mechanisms of succinate-mediated catabolite repression. Thus, some raffinose could be transported by the overproduced beta-galactoside transporters and cause the induction of alpha-galactoside utilization genes in the presence of both succinate and raffinose. This model is supported by the restoration of diauxie in a lacF lacR double mutant (lacF encodes a beta-galactoside transport protein) grown in medium containing succinate and raffinose. Biochemical support for the idea that succinate-mediated repression operates by preventing inducer accumulation also comes from uptake assays, which showed that cells grown in raffinose and exposed to succinate have a decreased rate of raffinose transport compared to control cells not exposed to succinate.

Raffinose content may influence cottonseed susceptibility to aflatoxin contamination

AbstractThe effect of the cotton storage trisaccharide raffinose and cottonseed storage protein (CSP) in combination on aflatoxin production by Aspergillus flavus was investigated. The ability of ground whole cottonseed and water‐extracted cottonseed meal to support fungal biosynthesis of aflatoxin also was assessed in vitro. Dose response data showed that utilization of raffinose as a sole carbon source supported growth and aflatoxin production by A. flavus. When raffinose was a carbon source and CSP was the sole nitrogen source, aflatoxin levels were stimulated up to fourfold above those in raffinose reference media. Results with ground whole cottonseed as a sole carbon/nitrogen source demonstrated the capacity for aflatoxin production in A. flavus cultures. Lipid extraction of ground whole seed severely reduced aflatoxigenesis potential; however, lipid extraction followed by water extraction of ground whole seed restored much of the aflatoxin biosynthetic potential, suggesting the presence of a water soluble inhibitory factor. Accessible carbon appeared to be the limiting resource in water‐extracted meal, as a raffinose supplement stimulated aflatoxin production. Either cottonseed storage lipid or raffinose was capable of providing accessible carbon to A. flavus. Raffinose and CSP in developing and mature cottonseed may predispose seed to potentially high levels of aflatoxin contamination by A. flavus upon seed infection.

Degradation of acharan sulfate and heparin by Bacteroides stercoris HJ-15, a human intestinal bacterium.

When glycosaminoglycan (GAG)-degrading enzymes were measured in normal human stool suspensions, all 5 tested different stools degraded titrable heparin and acharan sulfate. GAG-degrading bacteria were screened from the isolates of human stools. Among them, HJ-15 had the most potent activities of heparinases (GAGs-degrading enzymes). However, HJ-15 produced the enzyme even if in the media without heparin. Acharan sulfate lyase was induced by acharan sulfate and heparin. Heparinase production was also induced by these GAGs. These enzymes, acharan sulfate lyase and heparinase, were produced in exponential and stationary phase of HJ-15 growth, respectively. Optimal pHs of the acharan sulfate lyase and heparinase activities were 7.2 and 7.5, respectively. The biochemical properties of HJ-15 was similar to those of B. stercoris. However, difference from B. stercoris was utilization of raffinose. This HJ-15 also degraded chondroitin sulfates A and C.

Streptomyces turgidiscabies sp. nov.

A new bacterial species is described, for which the name Streptomyces turgidiscabies is proposed. This organism causes potato (Solanum tuberosum) scab in eastern Hokkaido, Japan; the lesions caused are distinctly erumpent. In culture, S. turgidiscabies is distinct from other scab-causing Streptomyces species, having flexuous spore chains and grey mass colour. The spores of this organism are cylindrical and smooth. Its cell walls contain the LL-diaminopimelic acid isomer, and its DNA G + C content is 71 mol%. S. turgidiscabies does not produce melanin or other diffusible pigments, does not grow on agar media at pH 4.0 or 37 degrees C, is positive for utilization of raffinose and inulin as a carbon source, and is sensitive to streptomycin (20 micrograms ml-1), penicillin G (10 IU ml-1), polymyxin B (15 micrograms ml-1), and thallium acetate (10 micrograms ml-1). The levels of DNA relatedness within S. turgidiscabies strains are high but relatedness between strains of this species and strains of S. acidiscabies, S. scabies, S. caviscabies, S. griseus, S. setonii and S. tendae are low. The type strain is SY9113T (= ATCC 700248T = IFO 16080T).

The multiple-sugar metabolism ( msm) gene cluster of Streptococcus mutans is transcribed as a single operon

The multiple-sugar metabolism ( msm) locus of Streptococcus mutans constitutes a non-PTS sugar uptake system responsible for the transport and utilization of raffinose, melibiose and isomaltotrioses. While previous studies have used polar mutations to suggest that these genes are co-transcribed, there has not been any direct evidence to support this. In this report we present direct evidence that the msm genes can be transcribed as a single operon.

The multiple-sugar metabolism (msm) gene cluster of Streptococcus mutans is transcribed as a single operon

The multiple-sugar metabolism (msm) locus of Streptococcus mutans constitutes a non-PTS sugar uptake system responsible for the transport and utilization of raffinose, melibiose and isomaltotrioses. While previous studies have used polar mutations to suggest that these genes are co-transcribed, there has not been evidence to support this. In this report we present direct evidence that the msm genes can be transcribed as a single operon.

Molecular evidence supporting the existence of two major groups in uropathogenic Escherichia coli.

Molecular methods allow an extremely fine strain typing that can be used to establish the population structure of bacterial species. This methodology has been used to characterize a collection of 74 uropathogenic Escherichia coli obtained from three hospitals located in geographically distant towns in Spain, some representatives of the ECOR collection and other reference strains. Genomic DNA was analyzed by RAPD (Random Amplified Polymorphic DNA) that can characterize a bacterial strain to the level of defining individual clones. The 16S rDNA-23S rDNA spacers were amplified by PCR and submitted to restriction analysis. Finally, the presence or absence of G adhesins in Escherichia coli as well as the type of adhesin (three types are known) have been shown by PCR amplification followed by digestion with restriction enzymes. As expected a wide diversity was shown by RAPD and identical patterns were only found in the case of strains isolated from the same individual, an obvious case of relapse. Analysis of the spacers' restriction patterns showed the presence of two markedly differentiated clusters that we have named alpha and beta. Both RAPD and spacer restriction patterns originated similar clusters of strains showing a consistency in the evolution of the global genome with the sequence variation of the ribosomal spacers. Furthermore, most of the strains having G-adhesin, with only a few exceptions, corresponded to the alpha rRNA spacer group. The two spacer types detected were also consistent with some phenotypic markers such as sucrose and raffinose utilization. The alpha and beta clusters could be intraspecific groups produced by partial sexual isolation or other barriers that are originating a divergent evolution.

Molecular evidence supporting the existence of two major groups in uropathogenic Escherichia coli

Molecular methods allow an extremely fine strain typing that can be used to establish the population structure of bacterial species. This methodology has been used to characterize a collection of 74 uropathogenic Escherichia coli obtained from three hospitals located in geographically distant towns in Spain, some representatives of the ECOR collection and other reference strains. Genomic DNA was analyzed by RAPD (Random Amplified Polymorphic DNA) that can characterize a bacterial strain to the level of defining individual clones. The 16S rDNA-23S rDNA spacers were amplified by PCR and submitted to restriction analysis. Finally, the presence or absence of G adhesins in Escherichia coli as well as the type of adhesin (three types are known) have been shown by PCR amplification followed by digestion with restriction enzymes. As expected a wide diversity was shown by RAPD and identical patterns were only found in the case of strains isolated from the same individual, an obvious case of relapse. Analysis of the spacers' restriction patterns showed the presence of two markedly differentiated clusters that we have named α and ß. Both RAPD and spacer restriction patterns originated similar clusters of strains showing a consistency in the evolution of the global genome with the sequence variation of the ribosomal spacers. Furthermore, most of the strains having G-adhesin, with only a few exceptions, corresponded to the α rRNA spacer group. The two spacer types detected were also consistent with some phenotypic markers such as sucrose and raffinose utilization. The α and β clusters could be intraspecific groups produced by partial sexual isolation or other barriers that are originating a divergent evolution.

Involvement of plasmids in saprophytic performance and sodium chloride tolerance of clover rhizobia W14-2 in vitro

Most information on the role of plasmids in the ecology of rhizobia in soil has been obtained using rhizobial derivatives lacking a single plasmid only. Rhizobium leguminosarum biovar trifolii strain W14-2 contains four plasmids (a, b, c, d). The role of plasmids in saprophytic characteristics and sodium chloride tolerance of W14-2 in vitro was investigated using a library of plasmid-cured W14-2 derivatives that includes derivatives containing only one plasmid and no plasmid, as well as complemented derivatives. Plasmid involvement in certain phenotypes such as swarming (plasmid a), utilization of glycerol (plasmid a), adonitol (plasmid b), rhamnose and sorbitol (plasmid c) was tracked to single plasmids and was not influenced significantly by the other plasmids present in the cell. Presence of plasmid b or c was necessary for the utilization of arabinose and raffinose, whereas presence of both plasmids b and d was required for tolerance to high levels of sodium chloride. The latter phenotype may correspond to tolerance to chloride toxicity or to osmotolerance. Plasmids influenced growth of the strain in liquid HP (peptone-yeast extract-tryptone), a complex medium that was investigated as it contains a wide range of carbon substrates. Positive contributions of plasmids a (shorter duration of the lag phase and increased maximum density of cells), b and c (shorter log-phase generation time) were shown. Plasmid d was detrimental to the strain in HP when it was the only plasmid present in the cell, especially regarding log-phase generation time. Similarly, the derivative with only plasmid d was more sensitive to sodium toxicity than any other derivative, including the plasmidless derivative. Interactions between plasmids influenced growth in HP broth and salt tolerance, and detrimental effects of plasmid d were little expressed if any when the cell contained one plasmid or more besides plasmid d. No plasmid-cured derivative grew better than the wild-type overall, suggesting that the extrachromosomal genome contributes to the saprophytic competence of W14-2 in soil.

Role of two operators in regulating the plasmid-borne raf operon of Escherichia coli

The plasmid-borne raf operon encodes functions required for the inducible uptake and utilization of raffinose in Escherichia coli K12. The expression of three structural genes for alpha-galactosidase (rafA), Raf permease (rafB) and sucrose hydrolase (rafD) is negatively controlled by the binding of RafR repressor (rafR) to two operator sites, O1 and O2, that flank the -35 sequence of the raf promoter, PA. In vitro, O1 and O2 are occupied on increasing the concentration of RafR, without detectable preference for one site or the other or any indication of cooperative binding. Nucleotide substitutions at positions 3, 4 or 5 in an operator half-site prevented repressor binding, supporting a model that postulates specific interactions of these base pairs with the recognition helix of RafR. To study the role of each operator site, we have compared by gel shift analysis the binding of purified RafR repressor to DNA fragments containing the original O1O2 configuration or mutant O1 or O2. When either one of the two operators was inactivated by site-directed mutagenesis, both O1 and O2 exhibited the same affinity for repressor and the same sensitivity to arrest of repressor binding by the natural inducer, melibiose. However, in the native O1O2 configuration, simultaneous binding of RafR to both operators was sterically hindered, leading to a 13-fold decrease in the intrinsic affinity of an operator site for repressor, once the other site had been occupied. To assess the role of each operator in vivo, rafA was used as a reporter gene. A 1200-fold repression (100%) was exerted by RafR binding to the native O1O2 configuration, whereas O2 alone exerted 45% and O1 alone 6% repression of rafA transcription. The differential effects of O1 versus O2 on transcription (despite matching affinities of O1 and O2 for repressor) suggest that positioning of the O2-repressor complex between the -35 and -10 signals is crucial for transcription control and that repressor binding to the upstream O1 serves to enhance this effect.

Genetic and biochemical characterization of a phosphatidylinositol-specific phospholipase C in Saccharomyces cerevisiae.

Hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) by phosphatidylinositol-specific phospholipase C (PI-PLC) generates two second messengers, inositol 1,4,5-trisphosphate and 1,2-diacylglycerol. The polymerase chain reaction was used to isolate a Saccharomyces cerevisiae gene (PLC1) that encodes a protein of 869 amino acids (designated Plc1p) that bears greatest resemblance to the delta isoforms of mammalian PI-PLC in terms of overall sequence similarity and domain arrangement. Plc1p contains the conserved X and Y domains found in all higher eukaryotic PI-PLCs (51 and 29% identity, respectively, to the corresponding domains of rat delta 1 PI-PLC) and also contains a presumptive Ca(2+)-binding site (an E-F hand motif). Plc1p, modified by in-frame insertion of a His6 tract and a c-myc epitope near its amino terminus, was overexpressed from the GAL1 promoter, partially purified by nickel chelate affinity chromatography, and shown to be an active PLC enzyme in vitro with properties similar to those of its mammalian counterparts. Plc1p activity was strictly Ca2+ dependent: at a high Ca2+ concentration (0.1 mM), the enzyme hydrolyzed PIP2 at a faster rate than phosphatidylinositol, and at a low Ca2+ concentration (0.5 microM), it hydrolyzed PIP2 exclusively. Cells carrying either of two different deletion-insertion mutations (plc1 delta 1::HIS3 and plc1 delta 2::LEU2) were viable but displayed several distinctive phenotypes, including temperature-sensitive growth (inviable above 35 degrees C), osmotic sensitivity, and defects in the utilization of galactose, raffinose, and glycerol at permissive temperatures (23 to 30 degrees C). The findings reported here suggest that hydrolysis of PIP2 in S. cerevisiae is required for a number of nutritional and stress-related responses.

Nonstructural carbohydrates in dormant and afterripened wild oat caryopses

Nonstructural carbohydrates were determined in both embryo and endosperm of dormant (nongerminating) and afterripened (germinating) intact caryopses of wild oat (Avena fatua L.). No changes in endosperm starch or soluble sugar were observed at the onset of germination (18 h). No changes in glucose, fructose, sucrose or starch within dormant or afterripened embryos correlated with onset of visual germination. In afterripened embryos, depletion of raffinose (18 h), stachyose (18 h) and galactose (24 h) was correlated with germination. In contrast, raffinose‐family oligosaccharide levels in dormant embryos remained constant for 7 days following imbibition. Germination of isolated dormant embryos on 88 mM galactose‐containing media was accompanied by decreased endogenous levels of raffinose and stachyose. Isolated embryos from dormant caryopses incorporated 14C from 14C‐fructose into both raffinose and stachyose during 24 h of imbibition. In contrast, no 14C incorporation into stachyose was observed in embryos from afterripened caryopses. No 14C incorporation into raffinose was observed at 18 and 24 h. When in vitro activities of α galactosidase were measured, no temporal differences between dormant or afterripened caryopses were detected in either embryo or endosperm tissue. Although the mechanism associated with differences in utilization of raffinose and stachyose is yet unidentified, alterations in raffinose‐family oligosaccharide metabolism in the embryo appear to be a unique prerequisite for afterripening‐induced germination.

IMP2, a nuclear gene controlling the mitochondrial dependence of galactose, maltose and raffinose utilization in Saccharomyces cerevisiae

The IMP2 gene of Saccharomyces cerevisiae is involved in the nucleo-mitochondrial control of maltose, galactose and raffinose utilization as shown by the inability of imp2 mutants to grow on these carbon sources in respiratory-deficient conditions or in the presence of ethidium bromide and erythromycin. The negative phenotype cannot be scored in the presence of inhibitors of respiration and oxidative phosphorylation, indicating that the role of the mitochondria in the utilization of the above-mentioned carbon sources in imp2 mutants is not at the energetical level. Mutations in the IMP2 gene also confer many phenotypic alterations in respiratory-sufficient conditions, e.g. leaky phenotype on oxidizable carbon sources, sensitivity to heat shock and sporulation deficiency. The IMP2 gene has been cloned, sequenced and disrupted. The phenotype of null imp2 mutants is indistinguishable from that of the originally isolated mutant.

New SNF genes, GAL11 and GRR1 affect SUC2 expression in Saccharomyces cerevisiae.

To identify new genes required for depression of the SUC2 (invertase) gene in Saccharomyces cerevisiae, we have isolated mutants with defects in raffinose utilization. In addition to mutations in SUC2 and previously identified SNF genes, we recovered recessive mutations that define four new complementation groups, designated snf7 through snf10. These mutations cause defects in the derepression of SUC2 in response to glucose limitation. We also recovered five alleles of gal11 and showed that a gal11 null mutation decreases SUC2 expression to 30% of the wild-type level. Finally, one of the mutants carries a grr1 allele that converts SUC2 from a glucose-inducible gene.

Nucleotide sequences and operon structure of plasmid-borne genes mediating uptake and utilization of raffinose in Escherichia coli

The plasmid-borne raf operon encodes functions required for inducible uptake and utilization of raffinose by Escherichia coli. Raf functions include active transport (Raf permease), alpha-galactosidase, and sucrose hydrolase, which are negatively controlled by the Raf repressor. We have defined the order and extent of the three structural genes, rafA, rafB, and rafD; these are contained in a 5,284-base-pair nucleotide sequence. By comparisons of derived primary structures with known subunit molecular weights and an N-terminal peptide sequence, rafA was assigned to alpha-galactosidase (708 amino acids), rafB was assigned to Raf permease (425 amino acids), and rafD was assigned to sucrose hydrolase (476 amino acids). Transcription was shown to initiate 13 nucleotides upstream of rafA; a putative promoter, a ribosome-binding site, and a transcription termination signal were identified. Striking similarities between Raf permease and lacY-encoded lactose permease, revealed by high sequence conservation (76%), overlapping substrate specificities, and similar transport kinetics, suggest a common origin of these transport systems. alpha-Galactosidase and sucrose hydrolase are not related to host enzymes but have their counterparts in other species. We propose a modular origin of the raf operon and discuss selective forces that favored the given gene organization also found in the E. coli lac operon.

Growth phase modulation of the productivity of α-galactosidase in budding yeast cultures

The complete utilization of raffinose and melibiose by Saccharomyces cerevisiae requires the synthesis and secretion of an α-galactosidase encoded by a single MEL1 gene. The influence of culture conditions on the productivity of α-galactosidase in batch growth of yeast cells has been investigated. A modulation of the MEL1 gene expression has been observed depending on growth condition, with a maximum of enzyme productivity during a transient physiological phase corresponding to the early stationary phase. Our experiments demonstrate that the highest α-galactosidase productivity observed in the early stationary phase of growth is not associated with a starvation condition, but it is coupled to a particular growth phase. The increase of specific activity of the enzyme occurs mainly for periplasmic and intracellular fractions while the amount of enzyme secreted in the medium remains fairly constant.