Role of Maltose Enzymes in Glycogen Synthesis by Escherichia coli

Abstract

Mutants with deletion mutations in the glg and mal gene clusters of Escherichia coli MC4100 were used to gain insight into glycogen and maltodextrin metabolism. Glycogen content, molecular mass, and branch chain distribution were analyzed in the wild type and in ⌬malP (encoding maltodextrin phosphorylase), ⌬malQ (encoding amylomaltase), ⌬glgA (encoding glycogen synthase), and ⌬glgA ⌬malP derivatives. The wild type showed increasing amounts of glycogen when grown on glucose, maltose, or maltodextrin. When strains were grown on maltose, the glycogen content was 20 times higher in the ⌬malP strain (0.97 mg/mg protein) than in the wild type (0.05 mg/mg protein). When strains were grown on glucose, the ⌬malP strain and the wild type had similar glycogen contents (0.04 mg/mg and 0.03 mg/mg protein, respectively). The ⌬malQ mutant did not grow on maltose but showed wild-type amounts of glycogen when grown on glucose, demonstrating the exclusive function of GlgA for glycogen synthesis in the absence of maltose metabolism. No glycogen was found in the ⌬glgA and ⌬glgA ⌬malP strains grown on glucose, but substantial amounts (0.18 and 1.0 mg/mg protein, respectively) were found when they were grown on maltodextrin. This demonstrates that the action of MalQ on maltose or maltodextrin can lead to the formation of glycogen and that MalP controls (inhibits) this pathway. In vitro, MalQ in the presence of GlgB (a branching enzyme) was able to form glycogen from maltose or linear maltodextrins. We propose a model of maltodextrin utilization for the formation of glycogen in the absence of glycogen synthase.The synthesis of glycogen in bacteria occurs when they are grown with limited nutrients but an abundance of a carbon source (33,34). Escherichia coli accumulates glycogen at levels of more than half of its cell mass under optimal conditions. The glycogen gene cluster in E. coli consists of two operons oriented in tandem, glgBX and glgCAP, encoding enzymes that synthesize and degrade glycogen (12). The encoded enzymes are a branching enzyme (glgB), a debranching enzyme (glgX), an ADP-glucose pyrophosphorylase (glgC), a glycogen synthase (glgA), and a glycogen phosphorylase (glgP). The polymerization of the ␣-1,4-linked glucosyl chain is mediated via the transfer of glucose from ADP-glucose by GlgA, the glycogen synthase, onto the nonreducing ends of linear dextrins that are subsequently branched (formation of ␣-1,6-glycosyl linkage) by GlgB, the branching enzyme. The expression of the glg gene cluster is complicated. It involves the global carbon storage regulator CsrA (2, 53), the cyclic AMP (cAMP)/catabolite gene activator protein (CAP) system (39), and the stringent response (38). In addition, the two-component regulatory system PhoP-PhoQ (29) connects the system to Mg 2ϩ levels, and even the phosphotransferase system appears to affect the glycogen phosphorylase involved in the degradation of glycogen (42,43). glgS, an additional gene involved in glycogen synthesis, is not part of the glg gene cluster. It is not essential for ...

Reaction Kinetics of Substrate Transglycosylation Catalyzed by TreX of Sulfolobus solfataricus and Effects on Glycogen Breakdown

“…Moreover, a limited dextrin containing a branch chain of DP4 is produced via the action of glycogen phosphorylase and is also conveniently studied in relation to mammalian debranching enzyme. The average branch chain lengths of glycogen in E. coli (28), S. solfataricus (35), and B. subtilis (36) are DP7 to -11, and the structures are expected to be similar to that of mammalian glycogen (DP13). Meléndez-Hevia et al (34) and Meléndez et al (37) developed a mathematical model of the mammalian glycogen molecule.…”

“…Cells were harvested by centrifugation (6,000 ϫ g) at 4°C for 20 min, washed with ice-cold water, and resuspended in 5 ml 50 mM sodium acetate (pH 4.5). Glycogen was isolated from this suspension and quantified as described by Park et al (28).…”

The α-Glucan Phosphorylase MalP of Corynebacterium glutamicum Is Subject to Transcriptional Regulation and Competitive Inhibition by ADP-Glucose

“…The glycogen synthesis pathway via the glgC-encoded ADP-glucose pyrophosphorylase and the glgA-encoded glycogen synthase is also the major route for glycogen synthesis in E. coli (2,75). However, also for glgC-and/or glgA-deficient E. coli strains, glycogen synthesis has been described (76)(77)(78): apart from the ADP-glucose pyrophosphorylase, additional, unknown enzymes for ADP-glucose synthesis are present in E. coli. Thus, glgC-deficient E. coli strains still synthesize small amounts of glycogen in cells grown on glucose (76,77).…”

“…Thus, glgC-deficient E. coli strains still synthesize small amounts of glycogen in cells grown on glucose (76,77). Deletion of glgA in E. coli abolishes glycogen synthesis in cultivations on glucose; however, glgA-deficient E. coli cells growing on maltose synthesize glycogen-like ␣-glucans from long maltodextrins formed by MalQ (78). Also, mycobacteria such as Mycobacterium tuberculosis and M. smegmatis synthesize ␣-glucans not only via the well-known GlgC-GlgA pathway but also by two further pathways (79).…”

Identification and Characterization of an Archaeal Kojibiose Catabolic Pathway in the Hyperthermophilic Pyrococcus sp. Strain ST04

“…Among these, maltose and maltodextrin uptake systems have been widely investigated (3,4). In Gramnegative bacteria, such as Escherichia coli, maltose is assimilated through an ATP-binding cassette (ABC) transporter system and intracellular amylases, including 4-␣-glucanotransferase, maltodextrin glucosidase, and maltodextrin phosphorylase (5,6). However, the Gram-positive bacterium Bacillus subtilis takes up maltose and maltodextrin using a phosphoenolpyruvate-dependent phosphotransferase system (PTS) mediated by a maltose-specific enzyme, IICB, and an ABC transporter containing a maltodextrinbinding protein (7,8).…”