Relationship between antifreeze protein and freezing resistance in Pseudomonas putida GR12-2

Following transposon Tn5 mutagenesis of the plant growth-promoting rhizobac terium Pseudomonas putida GR12-2, mutants that have different freeze-resist ant properties were selected. Five of the freeze-sensitive mutants, i.e. FS M-5, -6, -14, -29, and -41, secreted a lower amount of antifreeze protein-( AFP) into the culture broth compared with the wild-type. Among of these fiv e mutants, the three mutants (FSM-6, FSM-14, and FSM-41) that have the lowe st level of freezing resistance (4.0-6.0% survival) also produce AFP at low levels (0.5-0.9 mug/mL) compared with the wild-type (4.8 mug/mL). The anti freeze and ice-nucleating activities of the AFP from these three mutant str ains were similar to those of wild-type. Furthermore, the decreased freezin g resistance from three mutants could be partially restored by adding purif ied AFP to mutant cell suspensions. Freezing resistance of three mutants wa s found to increase in proportion to the addition of AFP up to a concentrat ion of 50 mug/mL. We conclude that accumulation of AFP is one component of the mechanism for freezing resistance in bacteria.