Expression of the Bacillus subtilis TasA signal peptide leads to cell death in Escherichia coli due to inefficient cleavage by LepB.

Bacillus subtilis has five type I signal peptidases, one of these, SipW, is an archaeal-like peptidase. SipW is expressed in an operon (tapA-sipW-tasA) and is responsible for removing the signal peptide from two proteins: TapA and TasA. It is unclear from the signal peptide sequence of TasA and TapA, why an archaeal-like signal peptidase is required for their processing. Bioinformatic analysis of TasA and TapA indicates that both contain highly similar signal peptide cleavage sites, both predicted to be cleaved by Escherichia coli signal peptidase I, LepB. We show that expressing full length TasA in E. coli is toxic and leads to cell death. To determine if this phenotype is due to the inability of the E. coli LepB to process the TasA signal peptide, we fused the TasA signal peptide and two amino acids of mature TasA (up to P2') to both maltose binding protein (MBP) and β-lactamase (Bla). We observed a defect in secretion, indicated by an abundance of unprocessed protein with both TasA-MBP and TasA-Bla fusions. A series of mutations in both TasA-MBP and TasA-Bla were made around the junction of the TasA signal peptide and the fusion protein. Both of these studies indicate that residues around the predicted TasA signal sequence cleavage site, particularly the sequence from P3 to P2', inhibit processing by LepB. The cell death observed when TasA and TasA signal sequence fusion proteins are expressed is likely due to the TasA signal peptide blocking LepB and thereby the general secretion pathway.
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