BACILLUS-SUBTILIS LEVANSUCRASE - THE EFFICIENCY OF THE 2ND STAGE OF SECRETION IS MODULATED BY EXTERNAL EFFECTORS ASSISTING FOLDING

We investigated whether the concentration of H+ or metal ions such as Ca2+, or both, on the external side of the cytoplasmic membrane is inv olved in coupling of folding and secretion of Bacillus subtilis levans ucrase by studying the modulation of each isolated event. In vitro at 30 degrees C, in the absence of Ca2+, the equilibrium between the unfo lded and the folded states of levansucrase was rapidly and totally dis placed toward the folded state by a small ph shift from 7.4 to 6.0. Ca 2+ (> 5 mM) acted as a catalyst of folding at ph greater than or equal to 7. In vivo pulse-chase experiments at 30 degrees C showed that, in the absence of Ca2+, the rate and the yield of the second step of lev ansucrase secretion were strongly dependent on the external ph. In aci dic growth medium (ph 5.8), secretion was efficient, In contrast, at p h greater than or equal to 7, the presence of Ca2+ was essential for s ecretion. In bacteria grown at high temperature (48 degrees C), both e xternal acidic ph and Ca2+ were required for efficient secretion. More over, a levansucrase variant altered in its calcium affinity was effic iently secreted only under acidic growth conditions. Depending on the culture conditions, the differences in H+ or Ca2+ concentrations which are maintained between the opposite sides of the energized cytoplasmi c membrane could be adequate to catalyse conformational transition whi ch could play a critical role in the second step of the protein releas e. These environmental parameters could also affect the yield of secre tion of some other secretory proteins, leading to the hypothesis that several different secretion mechanisms could coexist in B. subtilis.