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Rhizobium leguminosarum cells were separated into four distinct fractions by using density gradient centrifugation for the separation of the outer and cytoplasmic membranes and lysozyme-EDTA treatment of whole cells for the isolation of the periplasmic and cytoplasmic fractions. These methods allowed the subcellular localization of R. leguminosarum proteins.

Fractionation of Rhizobium leguminosarum cells into outer membrane, cytoplasmic membrane, periplasmic, and cytoplasmic components