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The analysis of Gluconacetobacter diazotrophicus exoproteome under continuous culture and fructans production. 1Crespo J.M., 1Molinari M.L., 1Boiardi J.L., 2Silva E. and 2Teixeira, K.R.S. 1 CINDEFI (UNLP; CCT-La Plata, CONICET), Buenos Aires, Argentina. 2 Embrapa Agrobiologia BR 465 Km 7 Seropédica, Rio de Janeiro, Brazil. The combination of proteomic techniques and defined growth conditions under continuous culture contributes for understanding the relationship between bacterial nutritional status and enzyme secretions to biotechnological application. This approach leads to determination of the role of only one variable during comparative proteomic. Gluconacetobacter diazotrophicus is acid-tolerant nitrogen fixing bacterium that was isolated from sugarcane crop. This bacterium tolerates high sucrose content because secretes an extracellular enzyme, levansucrase (LsdA) that hydrolyzes sucrose and also produce fructans, polymers of b(2→6) fructosyl units of low (FOS) or high (levans) molecular weight. They stimulate the growth of bifidobacteria, prevent colon cancer and reduce serum levels of cholesterol, phospholipid and triglyceride. Their application as sweetener is based on their calories-free and non-carcinogenic properties. Most of all, fructan productions in large-scale is of industrial interest and their role as prebiotic and to food safety is recognized by the U.S Food and Drug Administration (FDA). Here, we present a protocol that was modified to improve the quality of protein preparations for the exoproteome analysis from samples obtained from continuous growth of G. diazotrophicus PAL5. The bacterial growth was performed using 1L of LGIM modified medium in a 2 L chemostat unit and the dilution rate was adjusted to 0.05 h-1. The growth temperature was 30º C and the pH was automatically maintained. The dissolved oxygen (DO) was continuously measured and controlled according to the agitation speed. Different nutritional conditions have been tested: 1) under limited or excess of carbon source (sucrose 20 or 100 g/l); 2) under BNF conditions (O2% <2%) or N-excess (3 g/l (NH4)2SO4). During steady-state, the samples were taken and centrifuged at 16000 x g for 30 minutes centrifuged to obtain cellular and supernatants fractions and 0.2 mM of PMSF solution were added to each samples prior to storage. The exoproteome from each sample was evaluated by 2D analysis. After isoeletrofocusing the strips were subjected to the SDS-PAGE, then proteins spots were stained and analyzed using the Image Master v. 7 software. The 2D-protein analysis of supernatants showed that the use of ethanol precipitation and dialysis against mixed resin aqueous suspension previous to protein precipitation protocol improved the quality of protein preparation to the isoelectric focusing step. The protein extraction from cellular fraction was improved by CTAB precipitation for removal of insoluble cell debris, DNA and polysaccharides, resulting in reduction or elimination of horizontal streaking or incomplete focused spots. The analysis of the exoproteome present in the supernatants showed that the concentration of sucrose in the medium resulted in differential protein expression. In addition, a series of proteins of approximately 60 kDa was observed within pI range 5.0 6.0 when inputs of low sucrose concentration were supplied during continuous culture. According to previous work of Hernandez et. al. these spots probably correspond to LsdA isoforms. References: Hernández L., Ramirez R., Hormaza J. V., Madrazo J. and Arrieta J. (1999). Increase levansucrase production by a genetically modified Acetobacter diazotrophicus strain in shaking batch cultures. Letters in Applied Microbiology 28: 41-44. Acknowledgements: This study was partially supported by CBAB/CNPq and INCT FBN in Brazil and by CONICET and CABBIO in Argentina.