Kup and KimA: Two potassium uptake sistems regulated by c-di-Amp in Enterococcus faecalis JH2-2

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

Salta

Congreso; LV Reunión Anual de SAIB; 2019

Sociedad Argentina de Investigación en Bioquímica

Enterococcus faecalis is a natural commensal member of the human gut flora that belongs to the group of lactic acid bacteria (LAB). In recent years however, it has emerged as a clinical important opportunistic pathogen. A distinct trait in the physiology of these bacteria is the ability to persist and thrive in harsh environments, that include heat, acid, oxidative and hyperosmotic stress. Despite its controversial profile, E. faecalis are part of food products, either due to contamination or as part of starter, adjunct or non-starter cultures. Besides, ion homeostasis is a key factor for all living cells. Particularly, potassium (K+) is the most abundant cation in the cytosol, and its uptake is tightly regulated. Intracellular K+ is important for cellular metabolic processes such as gene expression, pH homeostasis, osmotic adaptation and enzymatic activity. A search for genes present in E. faecalis genome coding for proteins with high homology for K+ transporters revealed the presence of a Kup as well as a KimA homologue. To study the functional properties of the proteins encoded by both genes we used E.coli LB650 (∆kdpABC5 ∆trkH ∆trkG). This strain is a triple mutant for the main K+ transporter systems and, hence, it is unable to grow at low K+ concentrations. E. faecalis JH2-2 kup and kimA genes were individually cloned in plasmid pWH844 using E. coli LB650 as host, to check if their expression could restore growth in minimal salt media when no KCl is added. Both evidence growth with or without K+ supplementation. These results suggest that Kup and KimA are involved in the K+ transport in. E. faecalis. On the other hand, Kup and KimA are regulated by c-di-AMP in L. lactis and B. subtilis, respectively. c-di-AMP is a recently discovered second messenger molecule found in a wide range of bacteria and is the only that is essential for the bacteria that produce it. c-di-AMP plays important roles in the regulation of diverse cellular pathways, including K+ homeostasis. Once Kup and KimA were identified as K+ transporters, the next step was to analyze the impact of this metabolite on the activity of both proteins. A co-expression system was established in E. coli 2003. This strain is deficient in the three major K+ uptake systems (ΔkdpABC5 kupD1 ΔtrkA), and is not able to grow in minimal salt media at low K+ concentrations. Very importantly, E. coli lacks c-di-AMP synthesizing enzymes, for which the co-expression of a c-di-AMP synthesizing enzyme (CdaA) and Kup or KimA allows the analysis of the phenotypic effect of c-di-AMP on these transporters, without interference of host-synthesized c-di-AMP. Growth curves performed under low K+ concentrations confirmed that c-di-AMP has an inhibitory effect on both Kup and KimA.