ABSTRACTBacillus subtilisand its close relatives are widely used in industry for the Sec-dependent secretory production of proteins. Like other Gram-positive bacteria,B. subtilisdoes not possess SecB, a dedicated targeting chaperone that posttranslationally delivers exported proteins to the SecA component of the translocase. In the present study, we have implemented a functional SecB-dependent protein-targeting pathway intoB. subtilisby coexpressing SecB fromEscherichia colitogether with a SecA hybrid protein in which the carboxyl-terminal 32 amino acids of theB. subtilisSecA were replaced by the corresponding part of SecA fromE. coli.In vitropulldown experiments showed that, in contrast toB. subtilisSecA, the hybrid SecA protein gained the ability to efficiently bind toE. coliSecB, suggesting that the structural details of the extreme C-terminal region of SecA constitute a crucial SecB binding specificity determinant. Using a poorly exported mutant maltose binding protein (MalE11) and alkaline phosphatase (PhoA) as model proteins, we could demonstrate that the secretion of both proteins byB. subtiliswas significantly enhanced in the presence of the artificial protein targeting pathway. Mutations in SecB that do not influence its chaperone activity but prevent its interaction with SecA abolished the secretion stimulation of both proteins, demonstrating that the implemented pathway in fact critically depends on the SecB targeting function. From a biotechnological view, our results open up a new strategy for the improvement of Gram-positive bacterial host systems for the secretory production of heterologous proteins.
Bacterial Community Members Increase Bacillus subtilis Maintenance on the Roots of Arabidopsis thaliana