Characterization of F0.F1 ATPase mutants and analysis of their contribution to the acid tolerance mechanism of pneumococcus

CORTES PR; PIÑAS GE; ALBARRACIN ORIO AG; ECHENIQUE J

Cataratas Iguazu

Congreso; XL Reunión Anual de la Sociedad Argentina de Investigación Bioquímica y Biología Molecular; 2004

SAIB

We have previously reported an acid tolerance response (ATR) in pneumococcus, a main human pathogen. It has been described that F0.F1-ATPase plays a role in ATR in bacteria. In this work, our aim was to analyse the contribution of this enzyme to the induction of pneumococcal ATR. F0 F1 ATPase is constituted by several subunits codified by atp genes, but insertional mutants could not be obtained due to they are essential for viability. Point mutations on atpAC genes, coding for two subunits of F0 complex, have been described to be responsible for optochin resistance. With the purpose to obtain these mutants, we analyzed nine optochinresistant clinical strains. The atpABC genes were amplified individually from each strain, PCR products were transformed to an uncapsulated pneumococcal strain, and mutants were selected by optochin resistance. Once these mutants were obtained in the same genetic background, the ATR phenotype was examined and compared with wild-type strain. We found that modifications in the subunits c (V48L) and a (W206C) showed a higher lethal pH than wild-type strain. In contrast, another mutation in the subunit c (G47V) showed an increased tolerance to acidic stress. In this work, we present ATR phenotypes of these atp mutants that were not reported previously in bacteria. We suggest that F0.F1-ATPase is one of the main components of the ATR mechanism in pneumococcus, most likely by its H+ extrusion activity that controls the intracellular pH.