Maltose and maltotriose can be formed endogenously in Escherichia coli from glucose and glucose-1-phosphate independently of enzymes of the maltose system

Decker, Katja; Peist, Ralf; Reidl, Joachim; Kossmann, M; Brand, Bettina; Boos, Winfried

doi:10.1128/jb.175.17.5655-5665.1993

Cited by 70 publications

(77 citation statements)

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“…Previously, we had proposed the existence of a maltose/maltotriose phosphorylase that would form maltose and maltotriose by the reversible condensation starting with glucose-1-phosphate and free glucose (25). As we have shown in this publication, the increase of glucokinase activity (13-fold over the chromosomally encoded level) had a pronounced effect on mal gene expression, indicating the importance of free glucose.…”

Section: Discussionmentioning

confidence: 58%

“…(ii) Mutants in malQ encoding amylomaltase, the key enzyme in the degradation of maltose and maltodextrins, are constitutive provided that glycogen can be synthesized by the cell. This is due to the endogenous formation of maltotriose from glycogen (25). (iii) Mutations in malK encoding the ATP-hydrolyzing subunit of the system become constitutive for the expression of malK and malE due to the loss of the repressor function of MalK (15,26,41,57).…”

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confidence: 99%

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Molecular characterization of glucokinase from Escherichia coli K-12

et al. 1997

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glk, the structural gene for glucokinase of Escherichia coli, was cloned and sequenced. Overexpression of glk resulted in the synthesis of a cytoplasmic protein with a molecular weight of 35,000. The enzyme was purified, and its kinetic parameters were determined. Its K m values for glucose and ATP were 0.78 and 3.76 mM, respectively. Its V max was 158 U/mg of protein. A chromosomal glk-lacZ fusion was constructed and used to monitor glk expression. Under all conditions tested, only growth on glucose reduced the expression of glk by about 50%. A fruR mutation slightly increased the expression of glk-lacZ, whereas the overexpression of plasmid-encoded fruR ؉ weakly decreased expression. A FruR consensus binding motif was found 123 bp upstream of the potential transcriptional start site of glk. Overexpression of glk interfered with the expression of the maltose system. Repression was strongest in strains that exhibited constitutive mal gene expression due to endogenous induction and, in the absence of a functional MalK protein, the ATP-hydrolyzing subunit of the maltose transport system. It was least effective in wild-type strains growing on maltose or in strains constitutive for the maltose system due to a mutation in malT rendering the mal gene expression independent of inducer. This demonstrates that free internal glucose plays an essential role in the formation of the endogenous inducer of the maltose system.In Escherichia coli and most other bacteria, glucose is transported by the phosphoenolpyruvate:sugar phosphotransferase system (PTS) as glucose-6-phosphate (53), thus eliminating the need for glucokinase in the utilization of glucose. In contrast, the utilization of glucose-containing disaccharides such as lactose, maltose, or trehalose involves the formation of glucose inside the cell and requires its phosphorylation for the effective utilization of the disaccharides. Surprisingly, the presence of a glk mutation (22) does not appear to be a disadvantage in the utilization of these disaccharides. Severe reduction in growth is observed only when, in addition to having the glk mutation, the strain also lacks the ability to phosphorylate glucose via the PTS pathway (14, 59). Two different findings might be relevant for this phenomenon. In the first, glucose and galactose, the products of intracellular ␤-galactosidase action, have been found in large amounts outside the cell after the uptake of lactose, implying that growth on lactose is mediated via the uptake of the secreted products (PTS-mediated phosphorylation in the case of glucose) (35). In the second, internal phosphorylation of glucose by the PTS has been evoked (14). The findings indicate that the enzyme II Glc is also responsible for the utilization of internal glucose.As a consequence, the interest in E. coli glucokinase has been low. Glucokinase activity in E. coli was measured as early as 1953 (21), and MM6, an E. coli mutant defective in the utilization of glucose, was shown to contain normal amounts of glucokinase (4). In that study, the K m of...

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Section: Discussionmentioning

confidence: 58%

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confidence: 99%

Molecular characterization of glucokinase from Escherichia coli K-12

et al. 1997

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“…In this case, unusable glucose is also secreted and the remaining maltodextrins cannot support growth despite the presence of maltodextrin phosphorylase (12). It seems that for growth on glycolytic carbon sources, the cell needs the capacity to phosphorylate glucose, indicating at the same time the presence of a cytoplasmic activity hydrolyzing glucose 6-phosphate or glucose 1-phosphate to glucose (19). Figure 7 represents our present view of trehalose metabolism in E. coli.…”

Section: Discussionmentioning

confidence: 99%

Trehalose-6-phosphate hydrolase of Escherichia coli

1994

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The disaccharide trehalose acts as an osmoprotectant as well as a carbon source in Escherichia coli. At high osmolarity of the growth medium, the cells synthesize large amounts of trehalose internally as an osmoprotectant. However, they can also degrade trehalose as the sole source of carbon under both high-and low-osmolarity growth conditions. The modes of trehalose utilization are different under the two conditions and have to be well regulated (W. Boos, U. Ehmann, H. Forkl, W. Klein, M. Rimmele, and P. Postma, J. Bacteriol. 172:3450-3461, 1990). At low osmolarity, trehalose is transported via a trehalose-specific enzyme II of the phosphotransferase system, encoded by treB. The trehalose-6-phosphate formed internally is hydrolyzed to glucose and glucose 6-phosphate by the key enzyme of the system, trehalose-6-phosphate hydrolase, encoded by treC. We have cloned treC, contained in an operon with treB as the promoter-proximal gene. We have overproduced and purified the treC gene product and identified it as a protein consisting of a single polypeptide with an apparent molecular weight of 62,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme hydrolyzes trehalose-6-phosphate with a Km of 6 mM and a V.. of at least 5.5iimol of trehalose-6-phosphate hydrolyzed per min per mg of protein. The enzyme also very effectively hydrolyzes p-nitrophenyl-a-D-glucopyranoside, but it does not recognize trehalose, sucrose, maltose, isomaltose, or maltodextrins. treC was sequenced and found to encode a polypeptide with a calculated molecular weight of 63,781. The amino acid sequence deduced from the DNA sequence shows homology (50%6 identity) with those of oligo-1,6-glucosidases (sucrase-isomaltases) of Bacilus spp. but not with those of other disaccharide phosphate hydrolases. This report corrects our previous view on the function of the treC gene product as an amylotrehalase, which was based on the analysis of the metabolic products of trehalose metabolism in whole cells.The disaccharide trehalose serves as an osmoprotectant in many different organisms. Its two glucose molecules are linked 1-1 oa-glycosidically. Thus, the sugar is nonreducing and has the unique quality of maintaining the fluidity of membranes under conditions of dryness and desiccation (16,17). In Escherichia coli, trehalose is synthesized internally as an answer to osmotic stress (23,45,48). Among other, more prominent osmoprotectants such as glycine betaine or proline (11,36), trehalose can contribute up to 20% of the entire capacity for osmotic protection of the cell (21, 32). To synthesize the osmoprotectant trehalose at high osmolarity, UDP-glucose and glucose 6-phosphate are used to form trehalose-6-phosphate, regardless of the carbon source. Trehalose-6-phosphate is subsequently dephosphorylated to give free trehalose. Trehalose-6-phosphate synthase is encoded by otsA (osmotic trehalose synthesis), and trehalose-6-phosphate phosphatase is encoded by otsB. The two genes are localized at 42 min on the chromosome, otsB...

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“…However, unlike in E. coli, maltose could not be an acceptor of this 4-a-glucanotransferase if present in F. succinogenes. In E. coli mutants lacking MalQ, maltotriose is derived from the degradation of glycogen by the action of the glycogen-debranching enzyme GlgX (22,23). In F. succinogenes, MD3 might be produced as proposed in these E. coli mutants.…”

Section: Relationship Between Oligosaccharide and Glycogen Metabolismmentioning

confidence: 99%

Carbohydrate metabolism inFibrobacter succinogenes: What NMR tells us

Forano

Delort

Matulovà

2008

Microbial Ecology in Health and Disease

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Fibrobacter succinogenes is a major rumen fibrolytic bacterium found in high numbers when ruminants are fed cellulose-rich diets. It produces a very efficient fibrolytic system comprising numerous enzymes, but the organization and catalytic features of this system remain unclear. We were using nuclear magnetic resonance (NMR) to study carbohydrate metabolism by Fibrobacter spp. well before the term 'metabolomics' was coined. We first analyzed in detail the NMR spectra arising from resting cells of F. succinogenes provided with glucose, cellobiose or cellulose as a substrate. We were able to show carbohydrate cycling and reversibility of some steps. The use of 1D and 2D 1 H and 13 C NMR showed the synthesis by the cells of unexpected oligosaccharides: maltodextrins, maltodextrin-1-phosphate, and other glucose derivatives. We also showed that maltodextrins and maltodextrins-1-phosphate were synthesized and excreted out of the cell, and could originate from exogenous sugars or endogenous glycogen. According to the substrate and the physiological state of the bacterium (resting or growing cells), metabolic deviations toward the synthesis of these oligosaccharides were observed. We have also used liquid-and solid-state NMR to monitor wheat straw degradation by F. succinogenes. The originality of this NMR approach was to investigate the activity of an entire fibrolytic system on an intact complex substrate (wheat straw). No preferential degradation of amorphous versus crystalline cellulose was observed, nor of cellulose versus hemicelluloses. We showed for the first time a sequential activity of some enzymes of F. succinogenes S85 on wheat straw. In conclusion, our studies illustrate the utility of various NMR approaches to study and better understand sugar and polysaccharide metabolism in rumen bacteria.

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Maltose and maltotriose can be formed endogenously in Escherichia coli from glucose and glucose-1-phosphate independently of enzymes of the maltose system

Cited by 70 publications

References 41 publications

Molecular characterization of glucokinase from Escherichia coli K-12

Molecular characterization of glucokinase from Escherichia coli K-12

Trehalose-6-phosphate hydrolase of Escherichia coli

Carbohydrate metabolism inFibrobacter succinogenes: What NMR tells us

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