Comparison of subcellular fractionation methods for lactococcus lactis subsp. lactis and L. lactis subsp. cremoris

Cells of Lactococcus lactis subsp. lactis proved to be resistant to cell wall digestion by lysozyme using 0·6 M glycylglycine/10 mM MgCl 2 as stabilizing agent, this procedure having been described recently as suitable for subcellular fractionation of cells of L. lactis subsp. cremoris (Coolbear et al. (1992). Int. Dairy J., 2, 213-32). A procedure has now been developed for L. lactis subsp. lactis, based on the use of a combination of lysozyme and mutanolysin to digest the walls of cells stabilized in 24% sucrose/10 mM MgCl 2. Although the transfer of cell wall-depleted cells to hypotonic buffer resulted in lysis of most of the cells, a proportion of the cells remaining were permeable to small molecules (but not to proteins). The proportions of permeabilized cells, intact cells and cell wall-membrane complexes in the particulate fraction depended on both the lactococcal strain and the actual protocol used in the fractionation. Further, the concentration of N-acetylglucosamine in the subcellular fractions showed considerable variation between strains and the distribution of N-acetylglucosamine and another cell wall marker, rhamnose, in the fractions did not correlate. Lysylaminopeptidase activity was distributed between subcellular fractions in a similar manner to aldolase, but some evidence was obtained to suggest that a proportion of the activity may be associated with the cell membrane. For the four strains studied in detail (two strains each of L. lactis subsp. lactis and L. lactis subsp. cremoris), nearly half of the total proteinase and esterase activities were associated with the cell surface. The origin of the remainder of the activities was unclear.