Release of periplasmic enzymes fromRhizobium leguminosarum bvphaseoli bacteroids by lysozyme is enhanced by pretreatment of cells at low pH

Treatment ofRhizobium leguminosarum bvphaseoli bacteroids with 25 mM citrate buffer, pH 4.0, prior to treatment with 50 mM Tris, pH 8.0, containing 1 mM EDTA, 20% (wt/vol) sucrose, and 1 mg lysozyme/mL increased by two- to three-fold the release of the periplasmic marker enzymes phosphodiesterase and pyrophosphatase. Release of malate dehydrogenase, a cytoplasmic marker, was largely unaffected by the citrate pretreatment. The pretreatment was shown to be a pH effect, with a narrow optimum pH range. Above pH 4.0 the enhancement of release of periplasmic enzymes decreased sharply to almost no effect at pH 5.5, and below pH 4.0 release of malate dehydrogenase increased sharply. The effectiveness of pretreatment with low pH did not appear to be owing to increased entry of lysozyme into the periplasmic space, and an effect of low pH on the suceptibility of the peptidoglycan layer to hydrolysis is suggested.