Heat Resistance and Salt Hypersensitivity in Lactococcus lactis Due to Spontaneous Mutation ofllmg_1816(gdpP) Induced by High-Temperature Growth
ABSTRACTDuring construction of several gene deletion mutants inLactococcus lactisMG1363 which involved a high-temperature (37.5°C) incubation step, additional spontaneous mutations were observed which resulted in stable heat resistance and in some cases salt-hypersensitive phenotypes. Whole-genome sequencing of one strain which was both heat resistant and salt hypersensitive, followed by PCR and sequencing of four other mutants which shared these phenotypes, revealed independent mutations inllmg_1816in all cases. This gene encodes a membrane-bound stress signaling protein of the GdpP family, members of which exhibit cyclic dimeric AMP (c-di-AMP)-specific phosphodiesterase activity. Mutations were predicted to lead to single amino acid substitutions or protein truncations. An independentllmg_1816mutant (Δ1816), created using a suicide vector, also displayed heat resistance and salt hypersensitivity phenotypes which could be restored to wild-type levels following plasmid excision.L. lactisΔ1816 also displayed improved growth in response to sublethal concentrations of penicillin G. High-temperature incubation of a wild-type industrialL. lactisstrain also resulted in spontaneous mutation ofllmg_1816and heat-resistant and salt-hypersensitive phenotypes, suggesting that this is not a strain-specific phenomenon and that it is independent of a plasmid integration event. Acidification of milk by thellmg_1816-altered strain was inhibited by lower salt concentrations than the parent strain. This study demonstrates that spontaneous mutations can occur during high-temperature growth ofL. lactisand that inactivation ofllmg_1816leads to temperature resistance and salt hypersensitivity.