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

Department of Genetics, Groningen Biomolecular Sciences and Biotechnology Institute, PO Box 14, 9750 AA Haren, The Netherlands¹

Author for correspondence: Jan Maarten van Dijl. Tel: +31 50 363 3079. Fax: +31 50 363 2348. e-mail: J.M.VAN.DIJL{at}FARM.RUG.NL

The Gram-positive eubacterium Bacillus subtilis contains five chromosomally encoded type I signal peptidases (SPases) for the processing of secretory pre-proteins. Even though four of these SPases, denoted SipS, SipT, SipU and SipV, are homologous to the unique SPase I of Escherichia coli, they are structurally different from that enzyme, being almost half the size and containing one membrane anchor instead of two. To investigate whether the unique membrane anchor of Bacillus SPases is required for in vitro activity, soluble forms of SipS of B. subtilis, SipS of Bacillus amyloliquefaciens and SipC of the thermophile Bacillus caldolyticus were constructed. Of these three proteins, only a hexa-histidine-tagged soluble form of SipS of B. amyloliquefaciens could be isolated in significant quantities. This protein displayed optimal activity at pH 10, which is remarkable considering the fact that 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 been previously reported for the soluble form of the E. coli SPase, soluble SipS was active in the absence of added detergents. This observation can be explained by the fact that a highly hydrophobic surface domain of the E. coli SPase, implicated in detergent-binding, is absent from SipS.

Keywords: Bacillus subtilis, protein secretion, SipS, SipC

Abbreviations: Bam, Bacillus amyloliquefaciens; Bsu, Bacillus subtilis; CBB, Coomassie brilliant blue R; pre-A13i-Bla, pre(A13i)-ß-lactamase; pre-A2-AmyL, pre(A2)--amylase; SPase, signal peptidase

^a Present address: Department of Pharmaceutical Biology, University of Groningen, Antonius Deusinglaan 1, 9713 AV Groningen, The Netherlands.

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				M. L. van Roosmalen, J. D. H. Jongbloed, J.-Y. F. Dubois, G. Venema, S. Bron, and J. M. van Dijl Distinction between Major and Minor Bacillus Signal Peptidases Based on Phylogenetic and Structural Criteria J. Biol. Chem., June 29, 2001; 276(27): 25230 - 25235. [Abstract] [Full Text] [PDF]