Glycogen phosphorylase, the product of the glgP Gene, catalyzes glycogen breakdown by removing glucose units from the nonreducing ends in Escherichia coli.
ABSTRACT: To understand the biological function of bacterial glycogen phosphorylase (GlgP), we have produced and characterized Escherichia coli cells with null or altered glgP expression. glgP deletion mutants (DeltaglgP) totally lacked glycogen phosphorylase activity, indicating that all the enzymatic activity is dependent upon the glgP product. Moderate increases of glycogen phosphorylase activity were accompanied by marked reductions of the intracellular glycogen levels in cells cultured in the presence of glucose. In turn, both glycogen content and rates of glycogen accumulation in DeltaglgP cells were severalfold higher than those of wild-type cells. These defects correlated with the presence of longer external chains in the polysaccharide accumulated by DeltaglgP cells. The overall results thus show that GlgP catalyzes glycogen breakdown and affects glycogen structure by removing glucose units from the polysaccharide outer chains in E. coli.
Project description:The gene organization and transcription of the Agrobacterium glg operon differ from those in other bacteria. Agrobacterium tumefaciens A348 contains a 9.1-kb gene cluster harboring genes for glycogen metabolism. The nucleotide sequence and gene organization of a region containing ADP-glucose pyrophosphorylase (glgC), glycogen synthetase (glgA), and phosphoglucomutase (pgm) genes have been previously described (A. Uttaro and R. A. Ugalde, Gene 150:117-122, 1994). In this work we report that the glycogen phosphorylase (glgP) and branching enzyme (glgB) genes are located immediately upstream of this region. The complete nucleotide sequences of the glgP and glgB genes were obtained, and mutants were constructed by targeted insertional mutagenesis with a kanamycin cassette. Enzymatic assays and reverse transcription PCR carried out with the wild type and with glgP and glgB mutants, as well as primer extension experiments and beta-galactosidase fusions, revealed that this region containing five open reading frames (glgPBCA and pgm) is transcribed unidirectionally as a single operon under the control of a promoter located upstream of the glycogen phosphorylase gene (glgP). An alternative transcript was identified starting 168 bp upstream of an internal ATG start codon of the pgm gene, which is translated as a 71-amino-acid-shorter Pgm protein which complements in vivo a pgm mutant. This alternative transcript has a promoter with the motif TATCAAN5G, identified in octopine Ti plasmid as an autoinducible TraR promoter. This promoter is >200 times more efficient in A. tumefaciens than in Escherichia coli, as judged by the level of enzymatic activity of a lacZ-pgm fusion.
Project description:Glycogen metabolism contributes to energy storage and various physiological functions in some prokaryotes, including colonization persistence. A role for glycogen metabolism is proposed on the survival and fitness of Lactobacillus acidophilus, a probiotic microbe, in the human gastrointestinal environment. L.?acidophilus?NCFM possesses a glycogen metabolism (glg) operon consisting of glgBCDAP-amy-pgm genes. Expression of the glg operon and glycogen accumulation were carbon source- and growth phase-dependent, and were repressed by glucose. The highest intracellular glycogen content was observed in early log-phase cells grown on trehalose, which was followed by a drastic decrease of glycogen content prior to entering stationary phase. In raffinose-grown cells, however, glycogen accumulation gradually declined following early log phase and was maintained at stable levels throughout stationary phase. Raffinose also induced an overall higher temporal glg expression throughout growth compared with trehalose. Isogenic ?glgA (glycogen synthase) and ?glgB (glycogen-branching enzyme) mutants are glycogen-deficient and exhibited growth defects on raffinose. The latter observation suggests a reciprocal relationship between glycogen synthesis and raffinose metabolism. Deletion of glgB or glgP (glycogen phosphorylase) resulted in defective growth and increased bile sensitivity. The data indicate that glycogen metabolism is involved in growth maintenance, bile tolerance and complex carbohydrate utilization in L.?acidophilus.
Project description:Increasing evidence has shown that DNA methylation is involved in gene regulation in prokaryotes. However, there have been very limited reports about the role of DNA methylation in regulation of gene expression and physiological functions in cyanobacteria. In Synechocystis sp. PCC 6803, four genes on the plasmid pSYSX are predicted to encode the type I restriction-methylation system, slr6095 and slr6096 for the M subunit, slr6097 for the S subunit and slr6102 for the R subunit. Compared to the wild type, slr6095, slr6096, and slr6097 mutants lacked the GGm6AN7TTGG/CCAm6AN7TCC methylation in genomic DNA. Transcriptomic analysis indicated that 171 genes were reproducibly up- or down-regulated in all three mutants relative to the wild type. The changed expression of some genes, including sll1356 for glycogen phosphorylase (GlgP), was associated with the loss of GGm6AN7TTGG/CCAm6AN7TCC methylation in the coding regions or the upstream non-coding sequences. Inactivation of slr6095, slr6096, or slr6097 increased the expression of sll1356 and the GlgP activity but lowered the glycogen content. These results indicated that the DNA methylation by a type I RM system could alter the expression of certain genes and physiological functions in Synechocystis sp. PCC 6803.
Project description:?-Glucan phosphorylases contribute to degradation of glycogen and maltodextrins formed in the course of maltose metabolism in bacteria. Accordingly, bacterial ?-glucan phosphorylases are classified as either glycogen or maltodextrin phosphorylase, GlgP or MalP, respectively. GlgP and MalP enzymes follow the same catalytic mechanism, and thus their substrate spectra overlap; however, they differ in their regulation: GlgP genes are constitutively expressed and the enzymes are controlled on the activity level, whereas expression of MalP genes are transcriptionally controlled in response to the carbon source used for cultivation. We characterize here the modes of control of the ?-glucan phosphorylase MalP of the Gram-positive Corynebacterium glutamicum. In accordance to the proposed function of the malP gene product as MalP, we found transcription of malP to be regulated in response to the carbon source. Moreover, malP transcription is shown to depend on the growth phase and to occur independently of the cell glycogen content. Surprisingly, we also found MalP activity to be tightly regulated competitively by the presence of ADP-glucose, an intermediate of glycogen synthesis. Since the latter is considered a typical feature of GlgPs, we propose that C. glutamicum MalP acts as both maltodextrin and glycogen phosphorylase and, based on these findings, we question the current system for classification of bacterial ?-glucan phosphorylases.Bacterial ?-glucan phosphorylases have been classified conferring to their purpose as either glycogen or maltodextrin phosphorylases. We found transcription of malP in C. glutamicum to be regulated in response to the carbon source, which is recognized as typical for maltodextrin phosphorylases. Surprisingly, we also found MalP activity to be tightly regulated competitively by the presence of ADP-glucose, an intermediate of glycogen synthesis. The latter is considered a typical feature of GlgPs. These findings, taken together, suggest that C. glutamicum MalP is the first ?-glucan phosphorylase that does not fit into the current system for classification of bacterial ?-glucan phosphorylases and exemplifies the complex mechanisms underlying the control of glycogen content and maltose metabolism in this model organism.
Project description:1. The glycogen present in the liver of rat foetuses was labelled by injecting a trace amount of [6-(3)H]glucose into the mother at 19.5 days of gestation. The radioactivity incorporated in the glycogen 4h after the administration of the label was still present 38h later. A large proportion of this radioactivity was on the outer chains of the polysaccharide. These results indicate that there is normally almost no glycogen degradation in the foetal liver. In contrast, glycogen breakdown occurs very rapidly in the livers of foetuses whose mother is anaesthetized. 2. Glycogen synthetase is present in the liver at day 16 of gestation at a concentration as high as 30% of that in the adult, but essentially as an inactive (b) enzyme. The appearance of synthetase phosphatase between days 18 and 19 corresponds to that of synthetase a and to the beginning of glycogen synthesis. From day 19 to 21.5 the amount of synthetase a present in the foetal liver is just sufficient to account for the actual rate of glycogen deposition. 3. The content of total phosphorylase in the foetal liver increases continuously from day 16 to birth. However, a precise measurement of the a and b forms of the enzyme in the liver of non-anaesthetized foetuses is not possible. Taking the rate of glycogenolysis as an appropriate index of phosphorylase activity, we conclude that this enzyme is almost entirely in the inactive form in the foetal liver under normal conditions. 4. The accumulation of glycogen in the liver during late pregnancy may therefore be explained by a relatively slow rate of synthesis and a nearly total absence of degradation.
Project description:Glycogen is a highly branched polysaccharide that is widely present in all life domains. It has been identified in many bacterial species and functions as an important energy storage compound. In addition, it plays important roles in bacterial transmission, pathogenicity, and environmental viability. There are five essential enzymes (coding genes) directly involved in bacterial glycogen metabolism, which forms a single operon glgBXCAP with a suboperonic promoter in glgC gene in Escherichia coli. Currently, there is no comparative study of how the disruptions of the five glycogen metabolism genes influence bacterial phenotypes, such as growth rate, biofilm formation, and environmental survival, etc. In this study, we systematically and comparatively studied five E. coli single-gene mutants (?glgC, ?glgA, ?glgB, ?glgP, ?glgX) in terms of glycogen metabolism and explored their phenotype changes with a focus on environmental stress endurance, such as nutrient deprivation, low temperature, desiccation, and oxidation, etc. Biofilm formation in wild-type and mutant strains was also compared. E. coli wild-type stores the highest glycogen content after around 20-h culture while disruption of degradation genes (glgP, glgX) leads to continuous accumulation of glycogen. However, glycogen primary structure was abnormally changed in ?glgP and ?glgX. Meanwhile, increased accumulation of glycogen facilitates the growth of E. coli mutants but reduces glucose consumption in liquid culture and vice versa. Glycogen metabolism disruption also significantly and consistently increases biofilm formation in all the mutants. As for environmental stress endurance, glycogen over-accumulating mutants have enhanced starvation viability and reduced desiccation viability while all mutants showed decreased survival rate at low temperature. No consistent results were found for oxidative stress resistance in terms of glycogen metabolism disruptions, though ?glgA shows highest resistance toward oxidation with unknown mechanisms. In sum, single gene disruptions in glgBXCAP operon significantly influence bacterial growth and glucose consumption during culture. Accumulation and structure of intracellular glycogen were also significantly altered. In addition, we observed significant changes in E. coli environmental viabilities due to the deletions of certain genes in the operon. Further investigations shall be focused on the molecular mechanisms behind these phenotype changes.
Project description:We isolated a Tn5-induced Rhizobium tropici mutant that has enhanced capacity to oxidize N,N-dimethyl-p-phenylendiamine (DMPD) and therefore has enhanced respiration via cytochrome oxidase. The mutant had increased levels of the cytochromes c(1) and CycM and a small increase in the amount of cytochrome aa(3). In plant tests, the mutant increased the dry weight of Phaseolus vulgaris plants by 20 to 38% compared with the control strain, thus showing significantly enhanced symbiotic performance. The predicted product of the mutated gene is homologous to glycogen synthases from several bacteria, and the mutant lacked glycogen. The DNA sequence of the adjacent gene region revealed six genes predicted to encode products homologous to the following gene products from Escherichia coli: glycogen phosphorylase (glgP), glycogen branching enzyme (glgB), ADP glucose pyrophosphorylase (glgC), glycogen synthase (glgA), phosphoglucomutase (pgm), and glycogen debranching enzyme (glgX). All six genes are transcribed in the same direction, and analysis with lacZ gene fusions suggests that the first five genes are organized in one operon, although pgm appears to have an additional promoter; glgX is transcribed independently. Surprisingly, the glgA mutant had decreased levels of high-molecular-weight exopolysaccharide after growth on glucose, but levels were normal after growth on galactose. A deletion mutant was constructed in order to generate a nonpolar mutation in glgA. This mutant had a phenotype similar to that of the Tn5 mutant, indicating that the enhanced respiration and symbiotic nitrogen fixation and decreased exopolysaccharide were due to mutation of glgA and not to a polar effect on a downstream gene.
Project description:A chromosomal region of Bacillus stearothermophilus TRBE14 which contains genes for glycogen synthesis was cloned and sequenced. This region includes five open reading frames (glgBCDAP). It has already been demonstrated that glgB encodes branching enzyme (EC 184.108.40.206 [H. Takata et al., Appl. Environ. Microbiol. 60:3096-3104, 1994]). The putative GlgC (387 amino acids [aa]) and GlgD (343 aa) proteins are homologous to bacterial ADP-glucose pyrophosphorylase (AGP [EC 220.127.116.11]): the sequences share 42 to 70% and 20 to 30% identities with AGP, respectively. Purification of GlgC and GlgD indicated that AGP is an alpha2beta2-type heterotetrameric enzyme consisting of these two proteins. AGP did not seem to be an allosteric enzyme, although the activities of most bacterial AGPs are known to be allosterically controlled. GlgC protein had AGP activity without GlgD protein, but its activity was lower than that of the heterotetrameric enzyme. The GlgA (485 aa) and GlgP (798 aa) proteins were shown to be glycogen synthase (EC 18.104.22.168) and glycogen phosphorylase (EC 22.214.171.124), respectively. We constructed plasmids harboring these five genes (glgBCDAP) and assayed glycogen production by a strain carrying each of the derivative plasmids on which the genes were mutated one by one. Glycogen metabolism in B. stearothermophilus is discussed on the basis of these results.
Project description:The feasibility of using adenovirus as a vector for the introduction of glycogen phosphorylase activity into myocytes has been examined. We used the C2C12 myoblast cell line to assay the impact of phosphorylase gene transfer on myocyte glycogen metabolism and to reproduce in vitro the two strategies proposed for the treatment of muscle genetic diseases, myoblast transplantation and direct DNA delivery. In this study, a recombinant adenovirus containing the muscle glycogen phosphorylase cDNA transcribed from the cytomegalovirus promoter (AdCMV-MGP) was used to transduce both differentiating myoblasts and nondividing mature myotube cells. Muscle glycogen phosphorylase mRNA levels and total phosphorylase activity were increased in both cell types after viral treatment although more efficiently in the differentiated myotubes. The increase in phosphorylase activity was transient (15 days) in myoblasts whereas in myotubes higher levels of phosphorylase gene expression and activity were reached, which remained above control levels for the duration of the study (20 days). The introduction of muscle phosphorylase into myotubes enhanced their glycogenolytic capacity. AdCMV MGP-transduced myotubes had lower glycogen levels under basal conditions. In addition, these engineered cells showed more extensive glycogenolysis in response to both adrenaline, which stimulates glycogen phosphorylase phosphorylation, and carbonyl cyanide m-chlorophenylhydrazone, a metabolic uncoupler. In conclusion, transfer of the muscle glycogen phosphorylase cDNA into myotubes confers an enhanced and regulatable glycogenolytic capacity. Thus this system might be useful for delivery of muscle glycogen phosphorylase and restoration of glycogenolysis in muscle cells from patients with muscle phosphorylase deficiency (McArdle's disease).
Project description:1. Hepatocytes from starved rats or fed rats whose glycogen content was previously depleted by phlorrhizin or by glucagon injections, form glycogen at rapid rates when incubated with 10mM-glucose, gluconeogenic precursors (lactate, glycerol, fructose etc.) and glutamine. There is a net synthesis of glucose and glycogen. 14C from all three types of substrate is incorporated into glycogen, but the incorporation from glucose represents exchange of carbon atoms, rather than net incorporation. 14C incorporation does not serve to measure net glycogen synthesis from any one substrate. 2. With glucose as sole substrate net glucose uptake and glycogen deposition commences at concentrations of about 12--15mM. Glycogen synthesis increases with glucose concentrations attaining maximal values at 50--60mM, when it is similar to that obtained in the presence of 10mM glucose and lactate plus glutamine. 3. The activities of the active (a) and total (a+b) forms of glycogen synthase and phosphorylase were monitored concomitant with glycogen synthesis. Total synthase was not constant during a 1 h incubation period. Total and active synthase activity increased in parallel with glycogen synthesis. 4. Glycogen phosphorylase was assayed in two directions, by conversion of glycose 1-phosphate into glycogen and by the phosphorylation of glycogen. Total phosphorylase was assyed in the presence of AMP or after conversion into the phosphorylated form by phosphorylase kinase. Results obtained by the various methods were compared. Although the rates measured by the procedures differ, the pattern of change during incubation was much the same. Total phosphorylase was not constant. 5. The amounts of active and total phosphorylase were highest in the washed cell pellet. Incubation in an oxygenated medium, with or without substrates, caused a prompt and pronounced decline in the assayed amounts of active and total enzyme. There was no correlation between phosphorylase activity and glycogen synthesis from gluconeogenic substrates. With fructose, active and total phosphorylase activities increased during glycogen syntheses. 6. In glycogen synthesis from glucose as sole substrate there was a decline in phosphorylase activities with increased glucose concentration and increased rates of glycogen deposition. The decrease was marked in cells from fed rats. 7. To determine whether phosphorolysis and glycogen synthesis occur concurrently, glycogen was prelabelled with [2-3H,1-14C]-galactose. During subsequent glycogen deposition there was no loss of activity from glycogen in spite of high amounts of assayable active phosphorylase.