The maltodextrin system of Escherichia coli: metabolism and transport

Abstract

The maltose/maltodextrin regulon of Escherichia coli consists of 10 genes which encode a binding protein-dependent ABC transporter and four enzymes acting on maltodextrins. All mal genes are controlled by MalT, a transcriptional activator that is exclusively activated by maltotriose. By the action of amylomaltase, we prepared uniformly labeled [(14)C]maltodextrins from maltose up to maltoheptaose with identical specific radioactivities with respect to their glucosyl residues, which made it possible to quantitatively follow the rate of transport for each maltodextrin. Isogenic malQ mutants lacking maltodextrin phosphorylase (MalP) or maltodextrin glucosidase (MalZ) or both were constructed. The resulting in vivo pattern of maltodextrin metabolism was determined by analyzing accumulated [(14)C]maltodextrins. MalP(-) MalZ(+) strains degraded all dextrins to maltose, whereas MalP(+) MalZ(-) strains degraded them to maltotriose. The labeled dextrins were used to measure the rate of transport in the absence of cytoplasmic metabolism. Irrespective of the length of the dextrin, the rates of transport at a submicromolar concentration were similar for the maltodextrins when the rate was calculated per glucosyl residue, suggesting a novel mode for substrate translocation. Strains lacking MalQ and maltose transacetylase were tested for their ability to accumulate maltose. At 1.8 nM external maltose, the ratio of internal to external maltose concentration under equilibrium conditions reached 10(6) to 1 but declined at higher external maltose concentrations. The maximal internal level of maltose at increasing external maltose concentrations was around 100 mM. A strain lacking malQ, malP, and malZ as well as glycogen synthesis and in which maltodextrins are not chemically altered could be induced by external maltose as well as by all other maltodextrins, demonstrating the role of transport per se for induction.

FIG. 1.

TLC analysis of amylomaltase-mediated synthesis of ¹⁴C-labeled maltodextrins. The incubation mixture contained in 15 μl of 10 mM Tris-HCl, pH 7.5, crude dialyzed extract (final concentration, 1.5 mg protein/ml) of a strain containing overexpressed amylomaltase, 20 μM [¹⁴C]maltose, and the following unlabeled sugars at a 3 mM final concentration: in lanes 1 and 2, no addition; in lanes 3 and 4, maltose; in lanes 5 and 6, maltotriose; in lanes 7 and 8, maltotetraose; in lanes 9 and 10, maltopentaose; in lanes 11 and 12, maltohexaose; and in lanes 13 and 14, maltoheptaose. Lane M, flanking right, standard [¹⁴C]maltose; lane G, flanking left, standard [¹⁴C]glucose. Six-microliter samples were spotted on a TLC plate at 1 min after addition of the enzyme (odd-numbered lanes) and after 15 min (even-numbered lanes). Autoradiography was for 24 h.

FIG.2.

Fate of maltodextrins after accumulation in malQ strains lacking different maltodextrin-specific enzymes. The different strains were grown in NZA medium overnight. They were resuspended in MMA to an OD₅₇₈ of 0.5. Next, 0.015 to 0.02 μCi ¹⁴C-labeled maltodextrins (lane VII, maltoheptaose; lane VI, maltohexaose; lane V, maltopentaose; lane IV, maltotetraose; lane III, maltotriose; lane II, maltose) was added and the mixtures were incubated for 5 min. The pelleted bacteria were treated with TCA, and the supernatant was applied on TLC plates. Autoradiography was done for 1 week. The set at the right hand side of the figure (lanes 2 to 5) represents the corresponding dextrins without cells having the indicated numbers of glucosyl residues. Lane G, [¹⁴C]glucose standard; lane M, [¹⁴C]maltose. (A) Strain ST103 (MalQ⁻ MalP⁻ MalZ⁻); (B) strain ME469 (MalQ⁻ MalP⁻ MalZ⁺); (C) strain JH1 (MalQ⁻ MalZ⁻ MalP⁺).

FIG.2.

Fate of maltodextrins after accumulation in malQ strains lacking different maltodextrin-specific enzymes. The different strains were grown in NZA medium overnight. They were resuspended in MMA to an OD₅₇₈ of 0.5. Next, 0.015 to 0.02 μCi ¹⁴C-labeled maltodextrins (lane VII, maltoheptaose; lane VI, maltohexaose; lane V, maltopentaose; lane IV, maltotetraose; lane III, maltotriose; lane II, maltose) was added and the mixtures were incubated for 5 min. The pelleted bacteria were treated with TCA, and the supernatant was applied on TLC plates. Autoradiography was done for 1 week. The set at the right hand side of the figure (lanes 2 to 5) represents the corresponding dextrins without cells having the indicated numbers of glucosyl residues. Lane G, [¹⁴C]glucose standard; lane M, [¹⁴C]maltose. (A) Strain ST103 (MalQ⁻ MalP⁻ MalZ⁻); (B) strain ME469 (MalQ⁻ MalP⁻ MalZ⁺); (C) strain JH1 (MalQ⁻ MalZ⁻ MalP⁺).

FIG.2.

Fate of maltodextrins after accumulation in malQ strains lacking different maltodextrin-specific enzymes. The different strains were grown in NZA medium overnight. They were resuspended in MMA to an OD₅₇₈ of 0.5. Next, 0.015 to 0.02 μCi ¹⁴C-labeled maltodextrins (lane VII, maltoheptaose; lane VI, maltohexaose; lane V, maltopentaose; lane IV, maltotetraose; lane III, maltotriose; lane II, maltose) was added and the mixtures were incubated for 5 min. The pelleted bacteria were treated with TCA, and the supernatant was applied on TLC plates. Autoradiography was done for 1 week. The set at the right hand side of the figure (lanes 2 to 5) represents the corresponding dextrins without cells having the indicated numbers of glucosyl residues. Lane G, [¹⁴C]glucose standard; lane M, [¹⁴C]maltose. (A) Strain ST103 (MalQ⁻ MalP⁻ MalZ⁻); (B) strain ME469 (MalQ⁻ MalP⁻ MalZ⁺); (C) strain JH1 (MalQ⁻ MalZ⁻ MalP⁺).

FIG. 3.

Exit of [¹⁴C]maltose accumulated in CB47 by a plasmid encoding a sugar exit system. The culture was grown in MMA plus glycerol and resuspended in MMA without a carbon source to an OD₅₇₈ of 0.2. [¹⁴C]maltose at 0.14 μM (final concentration) was added and incubated for 15 min. The internal [¹⁴C]maltose concentration had then reached about 0.7 mM under equilibrium conditions. Unlabeled maltose (1 mM) was added at time zero to the medium, and samples were taken thereafter, filtered, and analyzed for radioactivity. Loss of radioactivity from the cell is given in cpm remaining inside the cells corresponding to a culture volume of 0.5 ml. ▪, strain CB47; •, strain CB47 harboring the plasmid-encoded uninduced levels of YabM, a sugar exit system.