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 involved in coupling of folding and secretion of Bacillus subtilis levansucrase by studying the modulation of each isolated event. In vitro at 30 degrees C, in the absence of Ca2+, the equilibrium between the unfolded and the folded states of levansucrase was rapidly and totally displaced toward the folded state by a small ph shift from 7.4 to 6.0. Ca2+ (> 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 levansucrase secretion were strongly dependent on the external ph. In acidic growth medium (ph 5.8), secretion was efficient, In contrast, at ph greater than or equal to 7, the presence of Ca2+ was essential for secretion. In bacteria grown at high temperature (48 degrees C), both external acidic ph and Ca2+ were required for efficient secretion. Moreover, a levansucrase variant altered in its calcium affinity was efficiently 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 cytoplasmic membrane could be adequate to catalyse conformational transition which could play a critical role in the second step of the protein release. These environmental parameters could also affect the yield of secretion of some other secretory proteins, leading to the hypothesis that several different secretion mechanisms could coexist in B. subtilis.