Selection of Protease-Positive and Protease-Negative Variants of Streptococcus cremoris

Protease-negative variants were shown to outcompete the wild-type strains of Streptococcus cremoris E(8), HP, and Wg(2) at pH values higher than 6.0 in milk. For S. cremoris E(8) this process was studied in more detail. At lower pH values the wild type had a selective advantage. This pH-dependent selection was not found in all media tested. The poor growth of the protease-negative variant at low pH was not due to lower internal pH values. By growing S. cremoris E(8) and Wg(2) in acidified milk (pH 5.9) the proteolytic activity of the cultures could be stabilized. In continuous cultures under amino acid limitation the wild type S. cremoris E(8) and HP strains had a selective advantage over the protease-negative variants at low dilution rates (D < 0.2) at all pH values of the medium. This was apparently due to a lower affinity-constant (K(s)) of the protease-positive variants for amino acids. Finally, a high fraction of protease-positive variants could be maintained in continuous cultures by using a growth medium with low concentrations of casein as a nitrogen source. At high dilution rates nearly all cells were protease positive.