Mutation of the response regulator RagA in Ensifer meliloti negatively affects survival under low pH

ALBICORO F. J.; MARTINI M. C.; QUIROGA M. P.; CENTRON D.; LAGARES A.; DEL PAPA M. F.

Córdoba

Congreso; XI SAMIGE, Córdoba, Argentina. 5 al 7 de agosto de 2015; 2015

SAMIGE

Ensifer meliloti, a root nodule bacterium, can establish symbiotic relationships with legumes resulting in atmospheric nitrogen being fixed to a form that can be utilized by the plant hosts. In its free-living state, this organism needs to overcome different unfavorable environmental conditions like soil acidity in order to survive, colonize the niche, reach the rhizophere and finally develop the nitrogen-fixing nodule in the legume roots. The so-called two-component systems (TCS) are part of the complex, diverse and broadly conserved strategy among bacteria for sensing and responding to external stimuli. TCS are typically comprised of a membrane-associated sensor kinase and a cytoplasmic response regulator. A better understanding of how this bacterium utilize TCS to sense acid stress condition and respond in consequence will help to elucidate the role of TCS in the rhizobial physiology. The aim of this work was to study the role of TCS involved in acid stress tolerance of E. meliloti. A collection of E. meliloti 2011 response regulator (RR) mutants were obtained either by a Tn5 transposon mutagenesis or by single cross-over method. Bacterial batch cultures, were performed in SG minimal medium under neutral (pH 7.0), sub-lethal (pH 5.6) or lethal acid conditions (pH 4.0). Differences were analyzed monitoring optic density at 600nm (OD 600 ) and colony-forming units (CFU) over time. In order to study the role of TCS in acid tolerance in E. meliloti, a collection of response regulators mutants was obtained. Growing rates were determined for each RR mutant as well as for the parental strain either in neutral (pH 7.0) or acid SG medium (pH 5.6). Measurement of OD 600 over time allowed us to determine which mutants exhibited a differential growth under stress conditions. This analysis permitted us to select one mutant (Smc02366 - , putative response regulator ragA) that showed a reduced growth rate in the stressed condition that was confirmed monitoring CFU over time. We also found that this mutant has lower capacity to tolerate a sudden shift to lethal acid condition comparing with the parental strain. There is no precedence in the literature about ragA participation in acid tolerance. We found that ragA would be essential for enhanced cell viability under lethal acid conditions. Efforts are therefore currently underway to determine the role of the components of this TCS in symbiosis and to explore the participation and regulation in a diverse range of abiotic stresses in E. meliloti.