Detergent-independent in vitro activity of a truncated Bacillus signal peptidase

The Cram-positive eubacterium Bacillus subtilis contains five chromosomally encoded type I signal peptidases (SPases) for the processing of secretory preproteins. Even though four of these SPases, denoted SipS, SipT, SipU and SipV, are homologous to the unique SPase I of Escherichia coli, they are s tructurally different from that enzyme, being almost half the size and cont aining one membrane anchor instead of two. To investigate whether the uniqu e membrane anchor of Bacillus SPases is required for in vitro activity solu ble forms of SipS of B. subtilis, SipS of Bacillus amyloliquefaciens and Si pC of the thermophile Bacillus caldolyticus were constructed. Of these thre e proteins, only a hexa-histidine-fagged soluble form of SipS of B. amyloli quefaciens could be isolated in significant quantities. This protein displa yed optimal activity at ph 10, which is remarkable considering the fact tha t the catalytic domain of SPases is located in an acidic environment at the outer surface of the membrane of living cells. Strikingly, in contrast to what has keen previously reported for the soluble form of the E. coli SPase , soluble SipS was active in the absence of added detergents. This observat ion can be explained by the fact that a highly hydrophobic surface domain o f the E. coli SPase, implicated in detergent-binding, is absent from SipS.