Iron uptake byBifidobacterium thermophilum protoplasts

Abstract

Protoplasts of Bifidobacterium thermophilum were prepared by a combination of lysozyme and protease digestion, and ferrous iron uptake studies were carried out. Little, if any, iron was internalized by the protoplasts, although large amounts of iron were bound to the protoplast surface. This binding was much greater than that of intact cells, which prefer to internalize iron by an energy-dependent process. It was also found that the binding of iron by protoplasts of cells grown in an iron-deficient medium was much more extensive than that of cells grown in an iron-sufficient medium. Soluble and particulate fractions of protoplasts were prepared by grinding them in a glass homogenizer, and the particulate fraction was also subjected to iron binding studies. The amount of iron bound was the same as that in intact protoplasts, indicating that the particulate fraction membrane fragments bound iron on their outer surface only. Nevertheless, when iron-preloaded cells were protoplasted and their surface cleared of iron, their particulate fraction contained considerable amounts of iron, indicating that the inner surface of the membranes is capable of binding iron as long as the cell is intact. The amount of iron so bound was dose-dependent on the amount of iron entering the cell. The failure of the outer and inner surface iron pools to mix was confirmed by the fact that when iron-preloaded protoplasts were incubated with additional iron, only the latter (surface-bound) was elutable with nonradioactive 2 mM FeSO4. It is concluded that increasing bifidobacterial iron load increases the amount of iron bound to the inner surface of the membrane; the procedure, which is effective in forming bifidobacterial protoplasts, destroys their iron transport mechanism while uncovering surface iron-binding sites; and that such iron-binding sites may be of significance in the cellular iron metabolism processes.

Binding of Ferric Iron to the Cell Walls and Membranes of Bifidobacterium thermophilum: Effect of Free Radicals

“…Protoplasting of B. thermophilum was carried out as previously described (Kot et al, 1993) using a mixture of lysozyme (Sigma Corp., St. Louis, MO, no. L-6876) and an Aspergillus oryzae protease (Sigma no.…”

“…The enzymes were present at 3 mg/mL concentration each, and the procedure was carried out for a total of 90 min. Protoplasts were further broken down into soluble and insoluble fractions by being suspended in water and ground in a glass homogenizer (Kot et al, 1993).…”

“…This cell suspension (20 mL) was maintained at 37 °C with gentle shaking with 5-mL aliquots being withdrawn at the 5-, 30-, 60-, and 120 min marks. Each cell sample thus withdrawn was cooled in an ice bath, centrifuged in the cold, washed once with the pH 6.5 buffer, and resuspended in the protoplasting solution (Kot et al, 1993). Following the protoplasting procedure, the reaction mixture was centrifuged and the solubilized and protoplast fractions were counted.…”