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Appl Environ Microbiol. 2012 Feb;78(3):651-9. doi: 10.1128/AEM.07209-11. Epub 2011 Nov 23.

Functional implementation of the posttranslational SecB-SecA protein-targeting pathway in Bacillus subtilis.

Author information

1
Institut für Bio- und Geowissenschaften, IBG-1: Biotechnologie, Forschungszentrum Jülich GmbH, Jülich, Germany.

Abstract

Bacillus subtilis and its close relatives are widely used in industry for the Sec-dependent secretory production of proteins. Like other Gram-positive bacteria, B. subtilis does 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 into B. subtilis by coexpressing SecB from Escherichia coli together with a SecA hybrid protein in which the carboxyl-terminal 32 amino acids of the B. subtilis SecA were replaced by the corresponding part of SecA from E. coli. In vitro pulldown experiments showed that, in contrast to B. subtilis SecA, the hybrid SecA protein gained the ability to efficiently bind to E. coli SecB, 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 by B. subtilis was 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.

PMID:
22113913
PMCID:
PMC3264108
DOI:
10.1128/AEM.07209-11
[Indexed for MEDLINE]
Free PMC Article

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