Transport of glucose by Bifidobacterium animalis subsp. lactis occurs via facilitated diffusion

Abstract

Two strains of Bifidobacterium animalis subsp. lactis were indistinguishable by several nucleic acid-based techniques; however, the type strain DSMZ 10140 was glucose utilization positive, while RB 4825, an industrially employed strain, was unable to grow rapidly on glucose as the principal carbon source. This difference was attributed to the presence of a low-affinity facilitated-diffusion glucose transporter identified in DSMZ 10140 but lacking in RB 4825. Uptake of D-[U-(14)C]glucose in DSMZ 10140 was stimulated by monovalent cations (ammonium, sodium, potassium, and lithium) and inhibited by divalent cations (calcium and magnesium). When competitor carbohydrates were included in the uptake assays, stereospecific inhibition was exhibited, with greater competition by methyl-beta-glucoside than methyl-alpha-glucoside. Significant inhibition (>30%) was observed with phloretin, an inhibitor of facilitated diffusion of glucose, whereas there was no inhibition by sodium fluoride, iodoacetate, sodium arsenate, sodium azide, 2,4-dinitrophenol, monensin, or valinomycin, which typically reduce energy-driven transport. Based on kinetic analyses, the mean values for K(t) and V(max) were 14.8 +/- 3.4 mM D-glucose and 0.13 +/- 0.03 micromol glucose/min/mg cell protein, respectively. Glucose uptake by several glucose-utilizing commercial strains of B. animalis subsp. lactis was also inhibited by phloretin, indicating the presence of facilitated diffusion glucose transporters in those strains. Since DSMZ 10140 has been previously reported to lack a functional glucose phosphoenolpyruvate phosphotransferase system, the glucose transporter identified here is responsible for much of the organism's glucose uptake.

FIG. 1.

PFGE of commercial and reference B. animalis subsp. lactis strains restricted using SpeI. Lanes 1 and 8, Lambda molecular weight marker; lanes 2 to 5, commercial strains of B. animalis subsp. lactis (lane 2, RB 0171; lane 3, RB 1280; lane 4, RB 1573; lane 5, RB 4825); lane 6, B. animalis subsp. lactis ATCC 27536; lane 7, B. animalis subsp. lactis DSMZ 10140.

FIG. 2.

Growth of B. animalis subsp. lactis strains DSMZ 10140 and RB 4825 in LL and LG media. Strains DSMZ 10140 and RB 4825 were grown in LL medium to mid- to late-log phase, harvested, washed with phosphate buffer, and then inoculated into LL broth, LG broth, or LG broth with 0.1% polysorbate 80 (LG+P80) and incubated at 37°C anaerobically. Growth was monitored as OD₆₀₀. Values represent the means of triplicate experiments; error bars represent standard deviations.

FIG. 3.

Utilization of lactose, product formation, and growth of B. animalis subsp. lactis DSMZ 10140 in LL medium. Strain DSMZ 10140 was grown in LL broth to mid- to late-log phase, harvested, washed with phosphate buffer, and then inoculated into LL broth and incubated at 37°C anaerobically. Growth was monitored as OD₆₀₀. Glucose, galactose, lactic acid, and acetic acid were monitored by HPLC. Data represent the mean of replicates; error bars represent standard deviations.

FIG. 4.

Effect of sodium chloride concentration on glucose uptake by B. animalis subsp. lactis DSMZ 10140. Glucose uptake was determined by growing DSMZ 10140 in LG broth to mid-log phase. Glucose uptake assays were performed in 0.05 M imidazole buffer with 1 mM glucose. Change in glucose uptake is expressed relative to the control, without added sodium chloride (mean value of 2.6 nmol/min/mg cell protein). Values represent the means of at least two replicate experiments; error bars represent standard deviations. Values with an asterisk are statistically different from the control without added sodium chloride (α = 0.05).

FIG. 5.

Effect of phloretin and phloridzin on glucose uptake by selected strains of B. animalis subsp. lactis. Glucose uptake was determined after growth in LG broth to mid-log phase. Glucose uptake assays were performed in 0.05 M imidazole buffer with 50 mM NaCl containing 1 mM glucose. Phloretin and phloridzin were prepared in ethanol and assayed with a 1 mM final concentration. An equivalent volume of ethanol was added to each control (1.25%). Change in glucose uptake is expressed relative to the control, without added inhibitor. Mean values of controls were 6.3 (DSMZ 10140), 6.4 (RB 1280), and 4.1 (RB 1573) nmol/min/mg cell protein from duplicate experiments; error bars represent standard deviations. Values with an asterisk are statistically different from the control without added inhibitor (α = 0.05).