SignificanceThe outer membrane (OM) excludes antibiotics such as vancomycin that kill gram-positive bacteria, and so is a major contributor to multidrug resistance in gram-negative bacteria. Yet, the OM is readily bypassed by protein bacteriocins, which are toxins released by bacteria to kill their neighbors during competition for resources. Discovered over 60 y ago, it has been a mystery how these proteins cross the OM to deliver their toxic payload. We have discovered how the bacteriocin pyocin S2 (pyoS2), which degrades DNA, enters Pseudomonas aeruginosa cells. PyoS2 tricks the iron transporter FpvAI into transporting it across the OM by a process that is remarkably similar to that used by its endogenous ligand, the siderophore ferripyoverdine.

Lectin-like bacteriocins consist of tandem monocot mannose-binding domains and display a genus-specific killing activity. Here we show that pyocin L1, a novel member of this family from Pseudomonas aeruginosa, targets susceptible strains of this species through recognition of the common polysaccharide antigen (CPA) of P. aeruginosa lipopolysaccharide that is predominantly a homopolymer of d-rhamnose. Structural and biophysical analyses show that recognition of CPA occurs through the C-terminal carbohydrate-binding domain of pyocin L1 and that this interaction is a prerequisite for bactericidal activity. Further to this, we show that the previously described lectin-like bacteriocin putidacin L1 shows a similar carbohydrate-binding specificity, indicating that oligosaccharides containing d-rhamnose and not d-mannose, as was previously thought, are the physiologically relevant ligands for this group of bacteriocins. The widespread inclusion of d-rhamnose in the lipopolysaccharide of members of the genus Pseudomonas explains the unusual genus-specific activity of the lectin-like bacteriocins.

Here we propose a mechanism of cell targeting and translocation for an S-type pyocin, pyocin SD2. Understanding and exploiting the mechanisms by which pyocins target, penetrate and kill Pseudomonas aeruginosa is a promising approach to antibiotic development.