CLASSICAL AND OMIC APPROACHES TO THE ANALYSIS OF ACID-STRESSED ALFALFA-NODULATING RHIZOBIA

DRAGHI,W., DEL PAPA, M.F., PISTORIO, M., LOZANO, M., MOLINARI, M.L., GIUSTI, M.A., BOIARDI, J.L., WATT, S., NIEHAUS, K., PÜHLER, A. Y LAGARES, A.

Universidad Austral. Pilar Buenos Aires-Argentina

Congreso; 1st Annual Iberoamerican PROTEOMICS Congress; 2007

Latinoamerican Human Proteome Organisation

The poor tolerance to the hydrogen ions of some rhizobia is considered among the main factors that restrict the development of N2-fixing symbioses with legumes in moderately acidic soils. This circumstance and the potential to extend current areas cultivated with legumes, both reinforce the necessity to understand the molecular rhizobial response to acidity towards its rational manipulation.To advance in the characterization of the rhizobial response to acidity at the proteome scale, and to investigate the relevance of differentially expressed markers for the wild-type acid tolerance; we set up N-limited continuous cultures of the model strain Sinorhizoboium meliloiti (Sme) 2011 at different pHs and studied their proteomes. In such experiments the extracellular pH was gradually decreased 0.2-0.5 units („b0.05) stepwise starting from a continuous culture at pH 7.4. Results showed that: strain Sme 2011 stopped growing upon reaching pHs lower than 6.1 in the chemostat (thus considered the pH limit ). 2D-gel electrophoresis-MALDI-TOF proteome analysis of strain 2011 sampled from the chemostat and from batch cultures at different pHs allowed for the identification of protein markers associated to the growth under each pH condition. We recognized 8 and 22 differentially expressed proteins associated to the acid and to the neutral condition, respectively. The up-regulated proteins under acidity included: Ppi and Tig (peptidylprolil isomerases, one of them ribosome-associated), SodB (superoxide dismutase), DegP1 (protease), TufA (translation factor), Mur (murein synthesis), and other proteins involved in the biosynthesis and/or transport of small molecules. Most of the acid-induced proteins were related to the support of housekeeping activities and to the recovery of protein structure/activities, denoting a reactive behavior of the cells against the operating stress condition. A very similar pattern of over/under expression was observed for the differential proteome markers at the transcriptome level, denoting a highly positive correlation between the proteome and transcriptome data under our experimental conditions. Finally, single Tn5 mutants affected in differential proteome markers showed in most cases more acid sensitive phenotypes. The collected data provided experimental support to screen and compare, in a final phenotypic screening, the functional contribution of each acid-induced/repressed marker to the growth of Sme under acidity.