Deciphering the metabolic details of L-lysine toxicity in cyanobacteria.

L-lysine (Lys) has been explored as a potential cyanobactericide due to its inhibitory effects on cyanobacterial growth at micromolar concentrations, comparable to many antibiotics. Here, we investigated the early metabolic and physiological responses of the model cyanobacterium Synechocystis sp. PCC 6803 to Lys exposure. Physiological analyses revealed cell enlargement, oxidative stress, and photosynthesis inhibition, leading to growth arrest. Metabolomic profiling indicated disruptions in peptidoglycan biosynthesis, evidenced by the accumulation of L-/D-alanine, meso-diaminopimelate, and D-Ala-D-Ala, suggesting interference with cell wall integrity. Furthermore, levels of energy metabolites and other amino acids, including tyrosine, tryptophan, valine, and iso-/leucine, were significantly altered, implying broader metabolic impacts of Lys toxicity. To explore potential resistance mechanisms, we used a CRISPRi-based genetic screen to identify key genes involved in relieving Lys toxicity. The Bgt permease system, responsible for basic amino acid uptake, was essential for acquiring Lys resistance, as a bgtA mutant exhibited normal growth on elevated Lys concentrations, thereby validating our CRISPRi screen. Additionally, UirR, a DNA-binding response regulator, and genes linked to c-di-AMP signaling seemed implicated in Lys metabolism. Deletion of the c-di-AMP synthase gene increased Lys sensitivity, supporting a role for c-di-AMP in cell wall homeostasis and osmotic stress regulation. Altogether, our findings explored the early metabolic responses and physiological consequences of Lys exposure in Synechocystis, demonstrating its effects on peptidoglycan biosynthesis, amino acid metabolism, and nucleotide biosynthesis. This, as well as the identification of key genetic factors contributing to Lys resistance, provides insights into cyanobacterial physiology and the potential application of Lys in bloom-control strategies.