Bacillus subtilis is a representative Gram-positive bacterium. In aerobic conditions, this bacterium can generate an electrochemical potential across the membrane with aerobic respiration. Here, we developed the patch clamp method to analyze the respiratory chain in B. subtilis. First, we prepared giant protoplasts (GPs) from B. subtilis cells. Electron micrographs and fluorescent micrographs revealed that GPs of B. subtilis had a vacuole-like structure and that the intravacuolar area was completely separated from the cytoplasmic area. Acidification of the interior of the isolated and purified vacuole-like structure, due to H(+) translocation after the addition of NADH, revealed that they consisted of everted cytoplasmic membranes. We called these giant provacuoles (GVs) and again applied the patch clamp technique. When NADH was added as an electron donor for the respiratory system, a significant NADH-induced current was observed. Inhibition of KCN and 2-heptyl-4-hydroxyquinoline-N-oxide (HQNO) demonstrated that this current is certainly due to aerobic respiration in B. subtilis. This is the first step for more detailed analyses of respiratory chain in B. subtilis, especially H(+) translocation mechanism.
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