From continuous culture to the proteome of Gluconacetobacter diazotrophicus.

MOLINARI M.L.; CRESPO J.M.; SILVA E.; TEIXEIRA K.R.S.

Buzios, Rio de Janeiro

Simposio; The 12th International Symposium on BNF with Non-Legumes; 2010

Embrapa Agrobiologia, Universidad Federal do Rio de Janeiro, Universidad Estadual Norte Fluminense (UENF), Institutos nacionais de ciéncia e tecnologia (inct)

The Gluconacetobacter diazotrophicus proteomes were studied under batch culture conditions. Nonetheless, this approach does not reflect the bacterial protein expression response to only one variable. G. diazotrophicus is an acid-tolerant endophyte, which associates and may contribute with fixed N to sugarcane and other non-leguminous crops. Although the interaction between the plant and endophytic bacteria is not fully understood, several plant growth promotion mechanisms have been attributed to it. The proteomic analyses of cellular and extracellular protein contents (exoproteome) under defined growth condition is currently one of the most promising tools to unravel the mechanisms involved during the plant-bacteria interaction. Besides, when combined to bacteria growth in continuous culture mode can provide samples that represent the influence of only one variable for comparative reliable proteomic analysis. However, factors such as excess of salts and exopolysaccharide productions can interfere to 2D-protein analysis from these sample types. Each continuous culture of G. diazotrophicus PAL5 was grown in a fermentor, containing 1L of LGIM modified medium, at 30º C and dilution rate adjusted to 0.05 h-1. To each treatment, the pH and dissolved oxygen were automatically monitored and controlled. Treatments evaluated consisted of continuous cultures that varied in the concentration of carbon source supply (sucrose 20 g/L or 100 g/L) in combination with two N-status. When the bacteria growth occurred at BNF, the dissolved oxygen was maintained at 2% maximum to simulate microaerophilic conditions. In contrast, when (NH4)2SO4 (3 g/L) was supplied the O2 level was maintained under air saturation during the growth. At steady-state, samples were collected, centrifuged to obtain cellular and supernatants fractions and 0.2 mM of PMSF solution were added to each samples prior to storage. Protein extraction protocols from cellular and extracellular fractions obtained under continuous culture conditions were developed. 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 the use of CTAB precipitation for removal of insoluble cell debris, DNA and polysaccharides, resulting in reduction or elimination of horizontal streaking or incomplete focused spots.