Abstract
The second messenger cyclic-di-AMP (c-di-AMP) is a signaling molecule widely present in gram-positive bacteria, where it regulates osmotic resistance by controlling potassium and compatible solute transport. Our previous studies using a Lactococcus cremoris model strain demonstrated that mutants with elevated c-di-AMP can overcome osmosensitivity through mutations enhancing potassium transporter activity. To identify additional mechanisms that enhance osmoresistance, we conducted a salt-resistance suppressor screen in an industrial L. cremoris strain. Using a spontaneous GdpP phosphodiesterase mutant with high c-di-AMP, we isolated salt-resistant suppressor mutants harboring six independent mutations in the khpB gene. These khpB mutants maintained elevated c-di-AMP levels comparable to the parental gdpP mutant. Inactivating khpB in wild-type and gdpP mutant laboratory L. cremoris strains similarly enhanced osmoresistance. KhpB (also known as EloR/Jag) is a putative RNA-binding protein, and its inactivation increased RNA transcript and protein expression of the glycine-betaine transporter BusAA-AB, elevating intracellular glycine-betaine uptake. Additionally, khpB disruption resulted in reduced cell size and enhanced secretion of native cell wall-degrading enzymes. Thus, KhpB likely acts as an indirect repressor of osmoresistance in L. cremoris by negatively regulating glycine-betaine transporter production.IMPORTANCELactococcus cremoris is a model lactic acid bacterium and an industrially valuable fermentation starter culture. Similar to other gram-positive bacteria, L. cremoris utilizes the nucleotide messenger c-di-AMP to manage responses to osmotic stress. A suppressor screen aimed at restoring salt resistance in a high c-di-AMP mutant identified several independent mutations within the khpB gene. Our results demonstrate that khpB disruption elevates intracellular glycine-betaine concentrations, a prominent osmoprotectant. Notably, khpB inactivation also reduced cell size and enhanced the secretion of native cell wall-degrading enzymes. This study thus reveals KhpB as a negative regulator of osmotic stress resistance in L. cremoris, thereby expanding our understanding of bacterial osmoadaptation mechanisms.