Glycogen formation in Corynebacterium glutamicum and role of ADP-glucose pyrophosphorylase.
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Glycogen is generally assumed to serve as a major reserve polysaccharide in bacteria. In this work, glycogen accumulation in the amino acid producer Corynebacterium glutamicum was characterized, expression of the C. glutamicum glgC gene, encoding the key enzyme in glycogen synthesis, ADP-glucose (ADP-Glc) pyrophosphorylase, was analysed, and the relevance of this enzyme for growth, survival, amino acid production and osmoprotection was investigated. C. glutamicum cells grown in medium containing the glycolytic substrates glucose, sucrose or fructose showed rapid glycogen accumulation (up to 90 mg per g dry weight) in the early exponential growth phase and degradation of the polymer when the sugar became limiting. In contrast, no glycogen was detected in cells grown on the gluconeogenic substrates acetate or lactate. In accordance with these results, the specific activity of ADP-Glc pyrophosphorylase was 20-fold higher in glucose-grown than in acetate- or lactate-grown cells. Expression analysis suggested that this carbon-source-dependent regulation might be only partly due to transcriptional control of the glgC gene. Inactivation of the chromosomal glgC gene led to the absence of ADP-Glc pyrophosphorylase activity, to a complete loss of intracellular glycogen in all media tested and to a distinct lag phase when the cells were inoculated in minimal medium containing 750 mM sodium chloride. However, the growth of C. glutamicum, its survival in the stationary phase and its glutamate and lysine production were not affected by glgC inactivation under either condition tested. These results indicate that intracellular glycogen formation is not essential for growth and survival of and amino acid production by C. glutamicum and that ADP-Glc pyrophosphorylase activity might be advantageous for fast adaptation of C. glutamicum to hyperosmotic stress. (+info)
Expression of alternansucrase in potato plants.
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Alternan, which consists of alternating alpha-(1-->3)/alpha-(1-->6)-linked glucosyl residues, was produced in potato tubers by expressing a mature alternansucrase (Asr) gene from Leuconostoc mesenteroides NRRL B-1355 in potato. Detection of alternan was performed by enzyme-linked immunosorbent assay in tuber juices, revealing a concentration between 0.3 and 1.2 mg g(-1) fresh wt. The Asr transcript levels correlated well with alternan accumulation in tuber juices. It appeared that the expression of sucrose-regulated starch-synthesizing genes (ADP-glucose pyrophosphorylase subunit S and granule-bound starch synthase I) was down-regulated. Despite this, the physico-chemical properties of the transgenic starches were unaltered. These results are compared to those obtained with other transgenic potato plants producing mutan [alpha-(1-->3)-linked glucosyl residues] and dextran [alpha-(1-->6)-linked glucosyl residues]. (+info)
Identification of regions critically affecting kinetics and allosteric regulation of the Escherichia coli ADP-glucose pyrophosphorylase by modeling and pentapeptide-scanning mutagenesis.
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ADP-glucose pyrophosphorylase (ADP-Glc PPase) is the enzyme responsible for the regulation of bacterial glycogen synthesis. To perform a structure-function relationship study of the Escherichia coli ADP-Glc PPase enzyme, we studied the effects of pentapeptide insertions at different positions in the enzyme and analyzed the results with a homology model. We randomly inserted 15 bp in a plasmid with the ADP-Glc PPase gene. We obtained 140 modified plasmids with single insertions of which 21 were in the coding region of the enzyme. Fourteen of them generated insertions of five amino acids, whereas the other seven created a stop codon and produced truncations. Correlation of ADP-Glc PPase activity to these modifications validated the enzyme model. Six of the insertions and one truncation produced enzymes with sufficient activity for the E. coli cells to synthesize glycogen and stain in the presence of iodine vapor. These were in regions away from the substrate site, whereas the mutants that did not stain had alterations in critical areas of the protein. The enzyme with a pentapeptide insertion between Leu(102) and Pro(103) was catalytically competent but insensitive to activation. We postulate this region as critical for the allosteric regulation of the enzyme, participating in the communication between the catalytic and regulatory domains. (+info)
The two AGPase subunits evolve at different rates in angiosperms, yet they are equally sensitive to activity-altering amino acid changes when expressed in bacteria.
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The rate of protein evolution is generally thought to reflect, at least in part, the proportion of amino acids within the protein that are needed for proper function. In the case of ADP-glucose pyrophosphorylase (AGPase), this premise led to the hypothesis that, because the AGPase small subunit is more conserved compared with the large subunit, a higher proportion of the amino acids of the small subunit are required for enzyme activity compared with the large subunit. Evolutionary analysis indicates that the AGPase small subunit has been subject to more intense purifying selection than the large subunit in the angiosperms. However, random mutagenesis and expression of the maize (Zea mays) endosperm AGPase in bacteria show that the two AGPase subunits are equally predisposed to enzyme activity-altering amino acid changes when expressed in one environment with a single complementary subunit. As an alternative hypothesis, we suggest that the small subunit exhibits more evolutionary constraints in planta than does the large subunit because it is less tissue specific and thus must form functional enzyme complexes with different large subunits. Independent approaches provide data consistent with this alternative hypothesis. (+info)
A sepal-expressed ADP-glucose pyrophosphorylase gene (NtAGP) is required for petal expansion growth in 'Xanthi' tobacco.
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In this study, a tobacco (Nicotiana tabacum 'Xanthi') ADP-glucose pyrophosphorylase cDNA (NtAGP) was isolated from a flower bud cDNA library and the role of NtAGP in the growth of the floral organ was characterized. The expression of NtAGP was high in the sepal, moderate in the carpel and stamen, and low in the petal tissues. NtAGP-antisense plants produced flowers with abnormal petal limbs due to the early termination of the expansion growth of the petal limbs between the corolla lobes. Microscopic observation of the limb region revealed that cell expansion was limited in NtAGP-antisense plants but that cell numbers remained unchanged. mRNA levels of NtAGP, ADP-glucose pyrophosphorylase activity, and starch content in the sepal tissues of NtAGP-antisense plants were reduced, resulting in significantly lower levels of sugars (sucrose, glucose, and fructose) in the petal limbs. The feeding of these sugars to flower buds of the NtAGP-antisense plants restored the expansion growth in the limb area between the corolla lobes. Expansion growth of the petal limb between the corolla lobes was severely arrested in 'Xanthi' flowers from which sepals were removed, indicating that sepal carbohydrates are essential for petal limb expansion growth. These results demonstrate that NtAGP plays a crucial role in the morphogenesis of petal limbs in 'Xanthi' through the synthesis of starch, which is the main carbohydrate source for expansion growth of petal limbs, in sepal tissues. (+info)
An Escherichia coli mutant producing a truncated inactive form of GlgC synthesizes glycogen: further evidences for the occurrence of various important sources of ADPglucose in enterobacteria.
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AC70R1-504 Escherichia coli mutants possess a glgC* gene with a nucleotide change resulting in a premature stop codon that renders a truncated, inactive form of GlgC. Cells over-expressing the wild type glgC, but not those over-expressing the AC70R1-504 glgC*, accumulated high ADPglucose and glycogen levels. AC70R1-504 mutants accumulated glycogen, whereas DeltaglgCAP deletion mutants lacking the whole glycogen biosynthetic machinery displayed a glycogen-less phenotype. AC70R1-504 cells with enhanced glycogen synthase activity accumulated high glycogen levels. By contrast, AC70R1-504 cells with high ADPG hydrolase activity accumulated low glycogen. These data further confirm that enterobacteria possess various sources of ADPglucose linked to glycogen biosynthesis. (+info)
Occurrence of more than one important source of ADPglucose linked to glycogen biosynthesis in Escherichia coli and Salmonella.
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To explore the possible occurrence of sources, other than GlgC, of ADPglucose linked to bacterial glycogen biosynthesis we characterized Escherichia coli and Salmonella DeltaglgCAP deletion mutants lacking the whole glycogen biosynthetic machinery. These mutants displayed the expected glycogen-less phenotype but accumulated ADPglucose. Importantly, DeltaglgCAP cells expressing the glycogen synthase encoding glgA gene accumulated glycogen. Protein chromatographic separation of crude extracts of DeltaglgCAP mutants and subsequent activity measurement analyses revealed that these cells possess various proteins catalyzing the conversion of glucose-1-phosphate into ADPglucose. Collectively these findings show that enterobacteria possess more than one important source of ADPglucose linked to glycogen biosynthesis. (+info)
The evolution of contact-dependent inhibition in non-growing populations of Escherichia coli.
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In the course of liquid culture, serial passage experiments with Escherichia coli K-12 bearing a mutator gene deletion (DeltamutS) we observed the evolution of strains that appeared to kill or inhibit the growth of the bacteria from where they were derived, their ancestors. We demonstrate that this inhibition occurs after the cells stop growing and requires physical contact between the evolved and ancestral bacteria. Thereby, it is referred to as stationary phase contact-dependent inhibition (SCDI). The evolution of this antagonistic relationship is not anticipated from existing theory and experiments of competition in mass (liquid) culture. Nevertheless, it occurred in the same way (parallel evolution) in the eight independent serial transfer cultures, through different single base substitutions in a gene in the glycogen synthesis pathway, glgC. We demonstrate that the observed mutations in glgC, which codes for ADP-glucose pyrophosphorylase, are responsible for both the ability of the evolved bacteria to inhibit or kill their ancestors and their immunity to that inhibition or killing. We present evidence that without additional evolution, mutator genes, or known mutations in glgC, other strains of E. coli K-12 are also capable of SCDI or sensitive to this inhibition. We interpret this, in part, as support for the generality of SCDI and also as suggesting that the glgC mutations responsible for the SCDI, which evolved in our experiments, may suppress the action of one or more genes responsible for the sensitivity of E. coli to SCDI. Using numerical solutions to a mathematical model and in vitro experiments, we explore the population dynamics of SCDI and postulate the conditions responsible for its evolution in mass culture. We conclude with a brief discussion of the potential ecological significance of SCDI and its possible utility for the development of antimicrobial agents, which unlike existing antibiotics, can kill or inhibit the growth of bacteria that are not growing. (+info)