Modular bacteriocins represent a major group of secreted protein toxins with a narrow spectrum of activity, involved in interference competition between Gram-negative bacteria. These antibacterial proteins include a domain for binding to the target cell and a toxin module at the carboxy terminus. Self-inhibition of producers is provided by coexpression of linked immunity genes that transiently inhibit the toxin’s activity through formation of bacteriocin-immunity complexes or by insertion in the inner membrane, depending on the type of toxin module. We demonstrate strain-specific inhibitory activity for PmnH, a Pseudomonas bacteriocin with an unprecedented dual-toxin architecture, hosting both a colicin M domain, potentially interfering with peptidoglycan synthesis, and a novel colicin N-type domain, a pore-forming module distinct from the colicin Ia-type domain in Pseudomonas aeruginosa pyocin S5. A downstream-linked gene product confers PmnH immunity upon susceptible strains. This protein, ImnH, has a transmembrane topology similar to that of Pseudomonas colicin M-like and pore-forming immunity proteins, although homology with either of these is essentially absent. The enhanced killing activity of PmnH under iron-limited growth conditions reflects parasitism of the ferrichrome-type transporter for entry into target cells, a strategy shown here to be used as well by monodomain colicin M-like bacteriocins from pseudomonads. The integration of a second type of toxin module in a bacteriocin gene could offer a competitive advantage against bacteria displaying immunity against only one of both toxic activities.

Bacteriocins related to colicin M, acting via cleavage of the cell wall precursor lipid II, have been characterized in γ- and β-proteobacteria. Depending on the species, immunity is provided by either an inner membrane-anchored periplasmic protein or by an integral membrane protein. In Pseudomonas however, the immunity partner of colicin M-like bacteriocins remains unknown. Based on an in silico analysis in pseudomonad genomes, we here identify a gene encoding a putative immunity partner that represents a novel type of integral membrane protein (PmiA, Pseudomonas colicin M-like immunity type A). By heterologous expression of pmiA genes in susceptible strains, we show that immunity to colicin M-like bacteriocins is indeed provided by the cognate PmiA. Sequence homology among PmiA proteins is essentially absent, except for a short motif with a conserved periplasm-exposed aspartate residue. However, PmiA's protective function is not abolished by changing this acidic residue to the uncharged alanine. Immunity by PmiAs appears promiscuous to the extent that PmiA homologs from a clade sharing <40% pairwise amino acid identity, equally provide protection against the bacteriocin linked to the original PmiA. This study shows that multiple immunity factors have evolved independently to silence lipid II-targeting enzymatic bacteriocins. Their relaxed bacteriocin immunization capacity contrasts to the strict specificity of immunity proteins shielding the enzymatic domain of nuclease bacteriocins. The nature of associated immune functions needs consideration when using such natural protein antibiotics or designing novel variants.