Expanding the toolbox: Novel class IIb microcins show activity against Gram-negative ESKAPE and plant pathogens

Interspecies interactions involving direct competition via bacteriocin production play a vital role in shaping ecological dynamics within microbial ecosystems. For instance, the ribosomally-produced siderophore bacteriocins, known as class IIb microcins, affect the colonization of host-associated pathogenicEnterobacteriaceaespecies. Notably, to date, only five of these antimicrobials have been identified, all derived from specificEscherichia coliandKlebsiella pneumoniaestrains. We hypothesized that class IIb microcin production extends beyond these specific compounds and organisms. With a customized informatics-driven approach, screening bacterial genomes in public databases with BLAST and manual curation, we have discovered twelve previously unknown class IIb microcins in seven additionalEnterobacteriaceaespecies, encompassing phytopathogens and environmental isolates. We introduce three novel clades of microcins (MccW, MccX, and MccZ), while also identifying eight new variants of the five known class IIb microcins. To validate their antimicrobial potential, we heterologously expressed these microcins inE. coliand demonstrated efficacy against a variety of bacterial isolates, including plant pathogens from the generaBrenneria, Gibbsiella, and Rahnella. Two newly discovered microcins exhibit activity against Gram-negative ESKAPE pathogens, i.e.Acinetobacter baumanniiorPseudomonas aeruginosa, providing the first evidence that class IIb microcins can target bacteria outside of theEnterobacteriaceaefamily. This study underscores that class IIb microcin genes are more prevalent in the microbial world than previously recognized and that synthetic hybrid microcins can be a viable tool to target clinically relevant drug-resistant pathogens. Our findings hold significant promise for the development of innovative engineered live biotherapeutic products tailored to combat these resilient bacteria.