C-DI-AMP AND POTASSIUM UPTAKE IN Enterococcus faecalis JH2-2

BLANCATO, VS; ACCIARRI, G; MAGNI, C; ESPARIZ, M

Congreso; LVI Reunión Anual de SAIB; 2020

Sociedad Argentina de Investigación Bioquímica y Biología Molecular

Nucleotide-based second messengers represent pivotal signal transduction mechanisms in all cell domains of life. Particularly in Firmicutes c-di-AMP has been recognized as an important messenger involved in essential cellular processes: such as wall synthesis, pathogenesis, osmotic response, and K+ uptake. In the Gram-positive bacterium, Enterococcus faecalis the c-di-AMP is produced by the di-adenylate cyclase CdaA and degraded by two phosphodiesterases, GdpP and PgpH. A clear link between c-di-AMP metabolism and virulence was established for E. faecalis. Regarding K+ uptake, it is known that is important for cellular metabolic processes such as pH homeostasis and osmotic adaptation. Besides, a distinct trait in the physiology of E. faecalis, compared to other lactic acid bacteria (LAB), is the ability to persist and thrive in harsh environments. In the last years, investigations directed the main role of c-di-AMP towards K+ metabolism. Due to all of this, we decided to study the K+ metabolism and its relation to c-di-AMP in E. faecalis. The mining of E. faecalis genome revealed the presence of the putative K+ transporters Kup, KimA, Ktr, and Kdp. Distribution of these transporters was not conserved among different strains of E. faecalis. Previous experiments showed that Kup and KimA are involved in the K+ transport in E. faecalis JH2-2. To analyze the impact of c-di-AMP on the activity of both proteins, a co-expression system was established in E. coli 2003 (defective K+ transporter strain, not able to grow in minimal media). E. coli also lacks c-di-AMP synthesizing enzymes, for which the co-expression of a CdaA and Kup or KimA allows the analysis of the phenotypic effect of c-di-AMP on these transporters. Growth curves confirmed that c-di-AMP has an inhibitory effect on Kup, but no effect has been seen on KimA. To assess the interaction between c-di-AMP and the potassium transporters in study, a differential radial capillary action of ligand assay (DRaCALA) was performed. Unfortunately, we were unable to prove the binding of c-di-AMP to Kup and KimA. To further examine the role of E. faecalis Kup, KimA, and KtrA proteins in K+ transport, we tested the growth of different K+ transporter mutants in E. faecalis JH2-2 in a low K+ medium at acidic, neutral, and alkaline starting pH. Whereas deletion of either kup or kimA alone and deletion of both genes had no impact on growth under all conditions analyzed, the ΔktrA mutant showed a defect on growth at pH 9. Notably the ΔkupΔktrA mutant exhibited a clear defect in growth in pH 9, low K+ medium. Hence, both E. faecalis Ktr and Kup systems were shown to be important for low-K+ growth under alkaline conditions. On the other hand, the fact that the ΔkupΔkimAΔktrA mutant strain could not be obtained so far suggests that at least one of the K+ uptake system studied must be active in E. faecalis JH2-2.