Intracellular proteomics analysis of Streptomyces sp. MC1 during sulfate and Cr(VI) simultaneous removal by Gel-Based and Gel-Free methods

BONILLA JO; CALLEGARI E; ESTEVEZ C; AMOROSO MJ; VILLEGAS LB

Córdoba

Congreso; XI Congreso Argentino de Microbiología General; 2015

SAMIGE

Streptomyces sp. MC1 has shown capacity for Cr(VI) reduction. The presence of sulfate ion mitigated the inhibitory effect of Cr(VI) on bacterial growth. Moreover, sulfate and chromium removal by this bacterium simultaneously increased when both ions were in the medium. The present work focused on differential protein expression from Streptomyces sp. MC1 under Cr(VI) stress in the presence of sulfate, using gel-based and gel-free methods or ?shotgun? analysis.The bacterium was grown in minimal medium containing glucose and 7.5 mM sulfate, with or without 20 ppm of Cr(VI) for 48h at 30°C and 180 rpm. Cells were chilled in liquid N2 and physically broken using mortar. The supernatants of cell lysates were used as protein samples.One aliquot of protein was concentrated by lyophilization for gel-free method and the other was concentrated by acetone precipitation for SDS-PAGE electrophoresis separation. Analysis of proteins samples mixtures were performed using 2 different approaches: 1) Identifying bands (SDS-PAGE) related to protein differential expression cut and gel digestion with trypsin, and 2) lyophilizing proteins dissolved in reaction buffer and trypsin digestion in solution. Tryptic peptides obtained were analyzed using 2D nano-Ultra Performance Liquid Chromatography, coupled to tandem mass spectrometry at SD Biomedical Research Infrastructure Network (SD BRIN) Proteomics Core Facility. Bioinformatics analysis for protein identification was performed by searching against Swiss Protein database with taxonomy filter for Streptomyces coelicolor A3, using Mascot server. ProteoIQ v2.8 program was used to organize the data after mass spectrometry analysis. The S. coelicolor A3 (as model of actinomycetes) genome was the primary selection for orthologue database since the sequences from Streptomyces sp. MC1 are not yet available.From use of these methods concludes: they are complementary to each other improving the number of proteins identified, majority of the intracellular proteins have values of isoelectric points in the range of pH 4-7, and the presence of sulfate and Cr(VI) in the culture medium increases the expression of many proteins. These proteins are involved in energy production, biosynthesis of macromolecules, cell division, chaperones, transcription and regulation of supercoiling, and DNA replication and repair. In addition, many dehydrogenases involved in redox processes and possibly involved in the reduction of Cr(VI) were expressed. The response of Streptomyces sp. MC1 against stress caused by Cr(VI), was not specific, but mechanisms of defense were activated based on the overexpression of proteins capable to cope the metal presence.This project demonstrated that combining gel electrophoresis with a gel-free approach improves the number of proteins identified, extends the available molecular weights and isoelectric points, and increases the dynamic range of the proteins, contributing to better quality and quantity of results.