More than a third of bacterial proteins are exported from the cytoplasm, of which over 90 percent occurs via the Sec pathway. Proteins exported via this pathway contain a short N-terminal signal peptide that is cleaved upon entry to the periplasm by signal peptidase I. There are two types of signal peptidases, a eukaryotic (ER-type) and a bacterial (P-type). Whilst the regions of the signal peptide have been extensively studied, less is known about how mature side residues influence protein export and cleavage by signal peptidase I. Recently, we have shown there is a bias against aromatic residues at the second amino acid position after signal peptide cleavage (P2’). When aromatic residues were added to maltose binding protein (MBP) at P2’, it leads to inefficient cleavage of the MBP signal peptide. However, one protein in Bacillus subtilis, TasA, contains an aromatic residue at this position and has its own dedicated, ancestral, ER-type signal peptidase. When the TasA signal peptide was fused to MBP and beta-lactamase (Bla), it resulted in either cell death (TasA-MBP) or increased sensitivity to beta-lactam antibiotics (TasA-Bla). Using SPR, we demonstrated that the TasA signal peptide can bind to LepB, the E. coli signal peptidase I enzyme, but is released up to 10-fold slower compared the MBP signal peptide. Further testing with short peptides revealed a motif, TWAAIE, that could inhibit LepB processing in an in vitro enzyme assay. Molecular modelling of the TasA signal peptide binding to LepB revealed that aromatic residues at P2 and P2’ sit close to the catalytic serine, potentially blocking its function. These results could aid the design of new inhibitors of signal peptidase I, an essential enzyme in bacteria, to help in the battle against antibiotic resistance.