Ó³»­´«Ã½

The impermeable outer membrane of is bypassed by antibacterial proteins known as S-type pyocins. Because of their properties, pyocins are investigated as a potential new class of antimicrobials against infections. Their production and modification, however, remain challenging. To address this limitation, we employed automated fast-flow peptide synthesis for the rapid production of a pyocin S2 import domain. The N-terminal domain sequence (PyS2) was synthesized in under 10 h and purified to yield milligram quantities of the desired product. To our knowledge, the 214 amino acid sequence of PyS2 is among the longest peptides produced from a "single-shot" synthesis, i.e., made in a single stepwise route without the use of ligation techniques. Biophysical characterization of the PyS2 with circular dichroism was consistent with the literature reports. Fluorescently labeled PyS2 binds to expressing the cognate ferripyoverdine receptor and is taken up into the periplasm. This selective uptake was validated with confocal and super resolution microscopy, flow cytometry, and fluorescence recovery after photobleaching. These modified, synthetic S-type pyocin domains can be used to probe import mechanisms of and leveraged to develop selective antimicrobial agents that bypass the outer membrane.