Differential protein synthesis of halotolerant microorganisms in presence of lithium chloride

MARTÍNEZ FABIANA; RAJAL VERÓNICA; IRAZUSTA VERÓNICA

Singapur

Congreso; 5th International Congress of Bacteriology and Applied Microbiology, 15th International Congress of Mycology and Eukaryotic Microbiology and 17th International Congress of Virology.; 2017

Objectives: We studied microorganisms isolated from El Salar del Hombre Muerto (salt flat in Catamarca, Argentina) to establish the mechanism they use to thrive in such an environment, composed mainly by sodium, lithium, and other metals. The main activity in the salt flat is the extraction of lithium, due to the high concentration of the brines, through natural evaporation processes benefiting from the high radiation and dryness of the zone. Our objective was to evaluate the differential synthesis of proteins that are involved in the interaction of the isolated bacteria with lithium chloride (LiCl).Methods: We used a defined liquid culture medium, composed by 10.0 g/L of glucose, 0.5 g/L of L-asparagine, 0.5 g/L of K2HPO4, 0.001 g/L of FeSO4.7H2O, and 0.020 g/L of MgSO4, without (MM, as control) and with the addition of 10, 20 and 30 g/L of LiCl (M10, M20 and M30), 2 and 4 M of sodium chloride (M2M and M4M respectively). The cultures were incubated at 30 °C and 200 rpm until exponential phase was reached and then the cells were harvested. Proteins were extracted using Urea buffer (Urea 8 M, Tris-HCl 25 mM at pH 8) and glass beads. Proteins were quantified using the Qubit kit. Mono dimensional denaturing polyacrylamide gel (1D SDS PAGE) and two-dimensional electrophoresis (2D SDS PAGE) were run. 2D analyses was carried out by means of an isoelectric focusing (7-cm strips, 3-11NL pH) and a SDS-PAGE (10%) for the first and second dimensions, respectively. Gels were coomassie-stained, scanned and analyzed. Two strains previously isolated and selected by their capacity to growth in the same medium supplemented with LiCl were used for this study. The first one was SA211, a yellow pigmented Gram positive coccoid bacteria, which grows in concentration of LiCl up to 50 g/L (M50), does not form biofilms (in the evaluated conditions) and its 16S sequence would identify it as a member of the genus Micrococcus. The second strain was SX139, a non-pigmented Gram positive rod, capable of forming biofilms and of growing in M50 which belongs to the genus Bacillus. Results: After 1D protein separation, it was clearly seen that the different media composition caused different protein profiles. For both strains, it was observed that MM, M2M and M4M were not useful to provide information since the bands were not well visible and yet, not comparable to any of the other conditions. The protein profile obtained in the situations M10 and M30 were comparable with each other and the differential synthesized proteins could be clearly seen. Only those proteins involved in the tolerance for a major amount of lithium would be shown, so those were the two situations chosen for comparison. Further, with the aim of studying these differences, 2D gels were carried out. 2D analysis revealed spots with differential intensities which were in agreement with the 1D study. Conclusion: As a conclusion, it is very clear that choosing the conditions that lead to a protein expression comparison is of major importance for a proper evaluation. Identifying these proteins will be the first step towards establishing the mechanism used by themicroorganisms that confers them the tolerance.