Studies have been undertaken on the effects of a number of parameters, including MgCl2 concentration, temperature, stabilizing buffer concentration and growth conditions on the response of cells of Lactococcus lactis subsp. cremoris strain E8 to subcellular fractionation procedures. Optimum stabilization of cells during partial cell wall digestion with lysozyme was obtained using 0.6 M glycylglycine, pH 7.5, containing 10 mM MgCl2. Concentrations of glycylglycine below 0.4 M severely reduced stabilization. Cooling of cell-wall-depleted cells below 20 degrees C caused considerable lysis; separation of these sensitive cells from solubilized cell wall material necessitated centrifugation at room temperature. Subsequent transfer of lysozyme-treated cells to ice-cold Tris-HCl, pH 7.5, achieved virtually complete lysis as determined using aldolase as a cytoplasmic marker. Increasing the MgCl2 concentration above 10 mM in the stabilizing buffer subsequently resulted in decreased lysis of protoplasts in the hypotonic buffer due, in part, to carry-over of MgCl2, MgCl2 could be substituted by CaCl2 with respect to stabilization, but proteinase distribution profiles between subcellular fractions were altered. KCl substituted only poorly for MgCl2. Inclusion of NaCl at even low concentrations in the hypotonic buffer decreased the levels of cell lysis. The distribution of two cell wall components, rhamnose and N-acetylglucosamine, between fractions did not correlate and responded differently to variations in MgCl2 concentration. Rhamnose remained almost entirely associated with the particulate material remaining after cell lysis. Two pools of N-acetylglucosamine were evident: a proportion of this monosaccharide could be readily released from the cell surface without loss of cell integrity, further release requiring more severe conditions and being accompanied by cell lysis. Cells grown in peptide-rich broth were more resistant to lysis after lysozyme treatment than when grown in reconstituted skim milk (RSM) and were almost completely resistant to lysis after mutanolysin treatment under the conditions used, whilst the RSM-grown cells were extremely susceptible to mutanolysin-induced lysis.
