The soluble pyocins S2 and S4 from Pseudomonas aeruginosa bind to the same FpvAI receptor

Soluble (S-type) pyocins are Pseudomonas aeruginosa bacteriocins that kill nonimmune P. aeruginosa cells by gaining entry via a specific receptor, which, in the case of pyocin S2, is the siderophore pyoverdine receptor FpvAI, and in the case of pyocin S3, FpvAII. The nucleic acid sequence at the positions 4327697–4327359 of P. aeruginosa PAO1 genome was not annotated, but it was predicted to encode the immunity gene of the flanking pyocin S4 gene (PA3866) based on our analysis of the genome sequence. Using RT-PCR, the expression of the immunity gene was detected, confirming the existence of an immunity gene overlapping the S4 pyocin gene. The PA3866 coding for pyocin S4 and the downstream gene coding for the immunity protein were cloned and expressed in Escherichia coli and the His-tagged S4 pyocin was obtained in pure form. Forty-three P. aeruginosa strains were typed via PCR to identify their ferripyoverdine receptor gene (fpvAI–III) and were tested for their sensitivity to pyocin S4. All S4-sensitive strains had the type I ferripyoverdine receptor fpvA gene. Some S4-resistant type I fpvA-positive strains were detected, but all of them had the S4 immunity gene, and, following the deletion of the immunity gene, became S4-sensitive. The fpvAI receptor gene was deleted in a S4-sensitive strain, and, as expected, the mutant became resistant to S4. The N-terminal receptor binding domain (RBD) of pyocin S2, which also uses the FpvAI receptor to enter the cell, was cloned in the pET-15b vector, and expressed in E. coli. When the purified RBD was mixed with pyocin S4 at different ratios, an inhibition of killing was observed, indicating that S2 RBD competes with the pyocin S4 for the binding to the FpvAI receptor. The S2 RBD was also shown to enhance the expression of the pvdA pyoverdine gene, suggesting that it, like pyoverdine, works via the known siderophore-mediated signalization pathway.