Differential protein synthesis of halotolerant microorganisms in presence of lithium chloride

FABIANA MARTÍNEZ; RAJAL, VERONICA BEATRIZ; IRAZUSTA, VERÓNICA

Singapore

Congreso; International Union of Microbiological Society (IUMS 2017), 15th International Congress of Bacteriology and Applied Microbiology, 15th International Congress of Mycology and Applied Eukaryotic Microbiology, 17th International Congress of Virology; 2017

Objectives: We studied microorganisms isolated from El Salar del Hombre Muerto (salt flat in Catamarca, Argentina) to establishthe mechanism they use to thrive in such an environment, composed mainly by sodium, lithium, and other metals. The main activityin the salt flat is the extraction of lithium, due to the high concentration of the brines, through natural evaporation processes benefitingfrom the high radiation and dryness of the zone. Our objective was to evaluate the differential synthesis of proteins that are involvedin 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 200rpm 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 anisoelectric focusing (7-cm strips, 3-11NL pH) and a SDS-PAGE (10%) for the first and second dimensions, respectively. Gels werecoomassie-stained, scanned and analyzed.Two strains previously isolated and selected by their capacity to growth in the same medium supplemented with LiCl were used forthis study. The first one was SA211, a yellow pigmented Gram positive coccoid bacteria, which grows in concentration of LiCl up to50 g/L (M50), does not form biofilms (in the evaluated conditions) and its 16S sequence would identify it as a member of the genusMicrococcus. The second strain was SX139, a non-pigmented Gram positive rod, capable of forming biofilms and of growing in M50which belongs to the genus Bacillus.Results: After 1D protein separation, it was clearly seen that the different media composition caused different protein profiles. Forboth strains, it was observed that MM, M2M and M4M were not useful to provide information since the bands were not well visibleand yet, not comparable to any of the other conditions. The protein profile obtained in the situations M10 and M30 were comparablewith each other and the differential synthesized proteins could be clearly seen. Only those proteins involved in the tolerance for amajor amount of lithium would be shown, so those were the two situations chosen for comparison. Further, with the aim of studyingthese differences, 2D gels were carried out. 2D analysis revealed spots with differential intensities which were in agreement with the1D study.Conclusion: As a conclusion, it is very clear that choosing the conditions that lead to a protein expression comparison is of majorimportance for a proper evaluation. Identifying these proteins will be the first step towards establishing the mechanism used by themicroorganisms that confers them the tolerance.