RT-qPCR analysis of dexB and galE gene expression of Streptococcus alactolyticus in Astragalus membranaceus fermentation.

Streptococcus alactolyticus strain FGM, isolated from chicken cecum, was used to increase the extract yield of polysaccharides during Astragalus membranaceus fermentation. It was previously demonstrated that polysaccharides from fermented A. membranaceus by S. alactolyticus had some similar properties to those from A. membranaceus in terms of its ability to help heal hepatic fibrosis in rats and modulate immunopotentiation of broiler chicken. However, methods to increase the yield of the polysaccharides during fermentation of A. membranaceus are not well understood. In this paper, we investigated the involvement of uridine diphosphate (UDP)-glucose 4-epimerase (galE) and glucan-1,6-α-glucosidase (dexB) during A. membranaceus fermentation through real-time reverse transcription quantitative PCR. The galE and dexB genes of S. alactolyticus were cloned by homology-based cloning and the genome walking method for the first time, and the 3D structure of dexB was analyzed by Swiss-PdbViewer 4.0.1 software. The expression of both the galE and dexB genes in A. membranaceus fermentation was studied using the determined ideal reference gene ldh for transcript normalization. The results showed that these two genes were both highly induced and peaked after 12 h of fermentation. The expression level of galE was stepwise increased from 48 to 72 h, while dexB transcripts were markedly increased at 60 h and decreased by 72 h. These data suggested that dexB and galE of S. alactolyticus strain FGM were involved in the regulation of A. membranaceus fermentation and they might play some roles in the increase of polysaccharides.

Efficient preparation of natural and synthetic galactosides with a recombinant beta-1,4-galactosyltransferase-/UDP-4'-gal epimerase fusion protein.

The numerous biological roles of LacNAc-based oligosaccharides have led to an increased demand for these structures for biological studies. In this report, an efficient route for the synthesis of beta-galactosides using a bacterial beta-4-galactosyltransferase/-UDP-4'-gal-epimerase fusion protein is described. The lgtB gene from Neisseria meningitidis and the galE gene from Streptococcus thermophilus were fused and cloned into an expression vector pCW. The fusion protein transfers galactose to a variety of different glucose- and glucosamine-containing acceptors, and utilizes either UDP-galactose or UDP-glucose as donor substrates. A crude lysate from Escherichia coli expressing the fusion protein is demonstrated to be sufficient for the efficient preparation of galactosylated oligosaccharides from inexpensive UDP-glucose in a multigram scale. Lysates containing the fusion protein are also found to be useful in the production of more complex oligosaccharides in coupled reaction mixtures, e.g., in the preparation of sialosides from N-acetylglucosamine. Thus, bacterially expressed fusion protein is well suited for the facile and economic preparation of natural oligosaccharides and synthetic derivatives based on the lactosamine core.

Glutamate and cyclic AMP regulate the expression of galactokinase in Mycobacterium smegmatis.

It was found that Mycobacterium smegmatis is unable to utilize galactose as the sole carbon source because the sugar alone cannot induce galactokinase. However, galactokinase was induced by glutamate alone, and was further stimulated by galactose. Rifampicin completely inhibited the glutamate-mediated expression of galK in both the absence and presence of galactose. Extracellular cAMP stimulated the expression of the enzyme only in the presence of glutamate plus galactose. The galK gene from M. smegmatis, including its upstream promoter region, was cloned in a plasmid in Escherichia coli. The expression of kinase from these clones in E. coli was dependent on cAMP and its receptor protein (CRP). The expression of UDP-galactose 4-epimerase was constitutive. This and other evidence suggests that the galK gene is not linked to galT and galE in the mycobacterial genome. In a glutamate-independent galactose-utilizing mutant (gin-1 mutant) of M. smegmatis, galK was expressed in the absence of both galactose and glutamate, while in the presence of galactose this expression was increased twofold in the absence of glutamate and fourfold in its presence. Extracellularly added cAMP reduced the expression of the enzyme in the presence of galactose plus glutamate nearly to the basal level. It is proposed that in M. smegmatis the galK gene is expressed from two different promoters; the expression from one promoter is dependent on glutamate but not on galactose and cAMP, while that from the other requires all three components. The role of galactose is possibly to derepress the latter promoter.

The Bacillus subtilis galE gene is essential in the presence of glucose and galactose.

Bacillus subtilis is unable to grow by consuming galactose because it is unable to transport it into the cell. The transcription of galE is not influenced by galactose but is repressed by glucose. Galactose is toxic for galE-negative bacteria because it results in elevated levels of metabolic intermediates. These negative effects are reduced in galK and galT mutants. Glucose is also toxic for galE-negative strains.

Amounts of proteins altered by mutations in the dnaA gene of Escherichia coli.

We identified proteins whose amounts were altered in a temperature-sensitive dnaA46 mutant of Escherichia coli. Proteins whose amounts were increased in the mutant were serine hydroxymethyltransferase, beta-ketoacyl [acyl carrier protein] synthase II, long-chain fatty acid transport protein, and UDP-glucose 4-epimerase, while the decreased ones were flagellin and D-ribose-binding protein. Transformation of the mutant with a plasmid containing the wild type dnaA gene complemented the phenotype. As pulse-labeling experiments revealed that the rates of synthesis of the proteins were altered in the mutant, DnaA protein may be involved in expression of these proteins.

Functional expression of uridine 5'-diphospho-glucose 4-epimerase (EC 5.1.3.2) from Arabidopsis thaliana in Saccharomyces cerevisiae and Escherichia coli.

It is our goal to investigate the biosynthesis of galactose-containing compounds in higher plants. Searching a database of expressed sequence tags, a cDNA from Arabidopsis thaliana (clone 108G20T7) with sequence similarity to UDP-glucose epimerase was identified and further analyzed. The 1356-bp-long cDNA included an open reading frame predicted to encode a 351 amino acid protein of 39 kDa. The presumed protein sequence showed a high degree of similarity to UDP-glucose epimerase sequences from bacteria, rat, and yeast. Complementation of the Saccharomyces cerevisiae gal1O mutant and expression of an active enzyme in Escherichia coli demonstrated that the cDNA encoded a functional UDP-glucose epimerase. The recombinant enzyme was purified to homogeneity. It showed a broad pH optimum of 7.0 to 9.5 and a Km of 0.11 mM. The UDP-glucose epimerase activity was not dependent on the addition of the cofactor NAD+ and was only moderately inhibited by high salt concentrations. Tissue-specific Northern analysis showed that the gene is expressed in all tissues of A. thaliana with highest expression levels in the stems and roots. Based on Southern analysis, there seems to be a single gene encoding UDP-glucose epimerase in A. thaliana. The cDNA analyzed during this study is the first known to encode a sugar-nucleotide modifying enzyme from higher plants. Its availability provides the means to investigate the role of UDP-glucose epimerase for the biosynthesis of UDP-galactose as precursor of galactolipids and cell wall polysaccharides.

Molecular cloning, characterization, and mapping of a full-length cDNA encoding human UDP-galactose 4'-epimerase.

Galactose metabolism in all organisms is catalyzed by three enzymatic steps: the galactokinase, galactose-1-phosphate uridyltransferase, and UDP galactose 4'-epimerase reactions. We report here the molecular cloning, characterization, and mapping of a full-length cDNA encoding human UDP-galactose 4'-epimerase (GALE). Our cDNA is 1488 bp long and matches the mRNA size of 1.5 kg detected in fibroblasts and lymphoblasts. The human GALE cDNA encodes a predicted protein of 348 amino acids with a molecular mass of 38,266. The human GALE enzyme is 87% identical to the rat protein, 53% identical to the homologous GAL10 protein from the yeast Kluyveromyces lactis, and 51% identical to the galE protein from the prokaryote Escherichia coli. This extraordinary degree of sequence identity has allowed us to build a homology model of the human protein based on the bacterial crystal structure. This predicted human structure is very similar to the E. coli galE enzyme, suggesting that both enzymes use similar mechanisms. The human gene encoding GALE maps, as expected, to a single locus on chromosome 1 and appears to be compact. The human GALE gene is structurally intact in 19 patients with epimerase-deficiency galactosemia, an inborn error of metabolism secondary to GALE deficiency. Therefore, we propose that this disorder is due to small mutations within the gene.

Rhizobium leguminosarum exoB mutants are deficient in the synthesis of UDP-glucose 4'-epimerase.

Rhizobium leguminosarum bv. viciae Exo- mutant strains RBL5523,exo7::Tn5,RBL5523,exo8::Tn5 and RBL5523,exo52::Tn5 are affected in nodulation and in the syntheses of lipopolysaccharide, capsular polysaccharide, and exocellular polysaccharide. These mutants were complemented for nodulation and for the syntheses of these polysaccharides by plasmid pMP2603. The gene in which these mutants are defective is functionally homologous to the exoB gene of Rhizobium meliloti. The repeating unit of the residual amounts of EPS still made by the exoB mutants of R. leguminosarum bv. viciae lacks galactose and the substituents attached to it. The R. leguminosarum bv. viciae and R. meliloti exoB mutants fail to synthesize active UDP-glucose 4'-epimerase.

Escherichia coli gal operon proteins made after prophage lambda induction.

Expression of the EScherichia coli gal operon under the control of the prophage lambda promoter pL leads to gross discoordinacy of gal expression. Expression of the most promoter-distal cistron galK is much greater than expression of the promoter-proximal cistron galE. We had previously shown that transcription of the gal operon is coordinate after prophage induction. A survey of protein synthesized after prophage induction indicated that lack of expression of galE is due to a failure of translation of the galE sequence in the pL-gal transcript. This failure of translation of the galE sequence may be due to extensive dyad symmetry present in the vicinity of the gal promoter region of the pL-gal transcript. This symmetry could result in a ribonucleic acid stem-loop structure, blocking the attachment of ribosomes at the Shine-Dalgarno sequence of galE. To test this model, strains bearing the IS1 or IS2 insertion, deletion, or new promoter mutation within the symmetrical region were constructed. The restoration of some galE expression after such disruptions of the symmetrical region indicated that the ribonucleic acid stem-loop structure did play a role in the discoordinate expression of gal from pL. However, failure to obtain galE expression coordinated with high levels of galK expression suggested that other components were involved, perhaps other symmetries between galE and the pL transcript.

Galactosamine is an inducer of Gal genes in Saccharomyces cerevisiae.

In yeast cells galactosamine in concentrations of 0.1 1M partially inhibits the synthesis of RNA but has little effect on the protein synthesis. In vivo and in vitro studies show that galactosamine is metabolized in yeast to UDP-N-acetylhexosamines but at a reduced rate, compared to the metabolism of galactose. The addition of galactosamine to growing yeast cells leads to the induction of the galactose pathway enzymes. Studies using different mutants in the galactose genes provide evidence that galactosamine is an inducer of the galactose structural genes in yeast. The same degree of induction of galactokinase and galactotransferase, found when galactose or galactosamine were used as inducers, supports the model of coordinated regulation in the expression of the structural genes for the galactose pathway enzymes in yeast.

Lactose metabolism by Streptococcus mutans: evidence for induction of the tagatose 6-phosphate pathway.

Growth on lactose by strains of Streptococcus mutans resulted in the induction of the lactose-phosphoenolpyruvate-phosphotransferase system, phospho-beta-galactosidase, and the enzymes of the tagatose 6-phosphate pathway.

Control of enzyme synthesis and stability during sporulation in Saccharomyces cerevisiae.

Studies were undertaken to understand the control of synthesis, stability and modification of UDP galactose epimerase and DNA-dependent RNA polymerase during sporulation of Saccharomyces cerevisiae. When a pre-induced culture of an inducible strain (wild type) is transferred to sporulation medium, the epimerase is inactivated to an undetectable level within 16 hours. Surprisingly, the addition of cycloheximide, a protein synthesis inhibitor, during sporulation stabilizes the epimerase activity. However, in a constitutive strain, the epimerase continues to be synthesized de novo during sporulation. Since the enzyme is synthesized during both vegatative growth and sporulation constitutively, the controls for synthesis of epimerase must be similar under these physiologically different conditions. After chromatography on DEAE Sephadex, there is no change observed in the elution patterns of RNA polymerase forms extracted from acetate growth vegetative cells, sporulating cells or from mature asci ; in all cases RNA polymerase consists of three forms, Ib, II and III. However, single spore suspension obtained from asci by treatment with zymolase contains a new form with chromatographic properties similar to those of form Ia. Our data suggests that form Ia may be a modification product of from Ib.

CO2 production from galactose in galactose-1-phosphate uridyl transferase-deficient Escherichia coli.

Escherichia coli K-12 deficient in galactose-1-phosphate uridyl transferase is capable of converting significant amounts of d-[1-(14)C]galactose to (14)CO(2), whereas strains deficient in other enzymes of the Leloir pathway cannot do so.

Activity, isoenzyme pattern, and synthesis of UDPglucose 4-epimerase during differtiation of Physarium polycephalum.

1. The specific activity of UDPglucose 4-epimerase (EC 5.1.3.2) increases by about 50% during the first 24 h of starvation-induced differentiation (spherulation) of Physarum polycephalum. 2. At all stages during differentiation, the enzyme activity is very sensitive to actinomycin-C and cycloheximide, inhibitors of transcription and translation, with a half life against cycloheximide of about 20 min (if added 12 h after the induction of differentiation). 3. The isoenzyme pattern, as revealed by isoelectric focusing in sucrose gradients, does not change during spherulation. One main band with a pI of 6.7, with a shoulder (pI 7.6) and a minor band (pI 6.0) was observed in extracts both from growing and differentiating cultures. 4. Density labelling experiments using deuterated amino acids with subsequent analysis by equilibrium density gradient sedimentation in 15-35% (w/w) metrizamide gradients revealed a rather slow rate of enzyme synthesis, which is in contrast to the observed high sensitivity against actinomycin-C and cycloheximide.