Preparation of giant spheroplasts and patch clamp

The standard procedure of giant spheroplast preparation from EC (Martinac et al., 1987) begins with growth in the presence of 0.06 mg/ml cephalexin, which blocks cell septation. Within 1.5–2 h, the growing but not dividing bacteria form 100–250-µm-long filaments. The filaments are transferred into a hypertonic buffer (1 M sucrose) and treated with 0.2 mg/ml lysozyme in the presence of 5 mM EDTA, which degrades the peptidoglycan layer within 5–10 min. As a result, filaments collapse into spheres 3–7 µm in diameter. The reaction is stopped by excess Mg²⁺ that terminates the effect of EDTA and activates DNase. Sedimentation through a one-step sucrose gradient separates the spheroplasts from the rest of the reaction mixture.

The procedure of PA giant spheroplast preparation was similar, but with two modifications. Instead of cephalexin, the PA filaments were grown in the presence of 0.2 mg/ml carbenicillin (1.5–2 h). Filaments were collected by low-speed centrifugation, resuspended in 1 M sucrose, and subjected to the “plasting” reaction in the presence of 10 mM EDTA and 0.6 mg/ml lysozyme for 15–20 min. Spheroplasts were separated from the debris by centrifugation through a one-step sucrose gradient, aliquoted, and stored at −80°C.

Borosilicate glass (Drummond 2-000-100) pipets 1–1.3 µm in diameter were used to form tight seals with the inner membrane. All recordings were done in excised inside-out patches in symmetrical 200 mM KCl, 10 mM CaCl₂, 90 mM MgCl₂ solution buffered by 10 mM HEPES, pH 7.2. Currents were recorded in voltage-clamp mode under preprogrammed mechanical stimuli combined from steps or ramps of negative pressure (suction) delivered from a modified HSPC-1 pressure clamp apparatus (ALA Scientific Instruments).