The influence of urea hydrolysis by urease on extracellular pH, on the intracellular pH and
on ATP synthesis was evaluated in the lactic acid bacterium Streptococcus thermophilus DSM
20617T. Urea hydrolysis to ammonium and carbon dioxide caused alkalinization of the cytoplasm,
the generation of a transmembranic pH gradient and a significant increase in ATP synthesis.
Urea-dependent ATP synthesis was completely abolished when cells were treated with
acetohydroxamic acid, a bacterial urease inhibitor or when cells of a recombinant urease-negative
mutant were used. The addition of the ionophores and ATPase inhibitor did not interfere with ATP
synthesis also at high concentration suggesting that a proton electrochemical potential and the
membrane ATPase were not involved in the urea-dependent ATP generation. The evaluation of
ATP synthesis compared to intra/extra-cellular pH variation in energetically discharged S.
thermophilus cells fed with lactose in presence or absence of urea suggested that the ureadependent ATP synthesis was generated by an accelerated glycolytic flux. The results obtained
together with the evaluation of lactose, glucose, lactic acid concentration, -galactosidase and
lactate dehydrogenase activity and in vivo
13
C NMR analysis suggested that the intracellular urea-
dependent alkalinization strongly increase lactose hydrolysis and the activity of glycolytic enzymes
thereby determining an increase of ATP synthesis. The overall data revealed new insights in the
regulation mechanisms involved in homolactic fermentation of S. thermophilus allowing to develop
new strategies to increase the yield in biomass production.
Growth competition experiments carried out in laboratory media and in milk between wildtype and urease-negative mutant revealed that urea hydrolysis, the related increase of lactose
consumption, and the improvement of glycolytic-dependent ATP generation strongly increased the
fitness of milk colonization of S. thermophilus.