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, have been observed to affect the colonization of pathogenic Enterobacteriaceae species within hosts. Notably, to date, only five of these antimicrobials have been identified and all were derived from specific Escherichia coli and Klebsiella pneumoniae strains. We hypothesized that class IIb microcin production extends beyond these specific compounds and organisms. By employing an informatics-driven approach, screening bacterial genomes in publicly accessible databases, we have discovered a total of twelve previously unknown class IIb microcins. Our investigation unveiled that these microcins are harbored within a diverse array of Enterobacteriaceae species, 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 previously known ones. To validate their antimicrobial potential, we heterologously expressed these microcins, along with their immunity peptides, in E. coli and unequivocally demonstrated their efficacy against a variety of bacterial isolates, including plant pathogens like Gibbsiella species and Rahnella victoriana . Remarkably, two of these newly discovered class IIb microcins exhibit activity against gram-negative ESKAPE pathogens, such as Acinetobacter baumannii or Pseudomonas aeruginosa providing the first evidence that class IIb microcins can target bacteria outside of the Enterobacteriaceae family. Our findings hold significant promise for the development of innovative live biotherapeutic products tailored to combat these resilient bacteria and underscore the notion that class IIb microcins are more prevalent and more broad-spectrum in the natural microbial world than previously recognized. ### Competing Interest Statement V.B. receives support from a sponsored research agreement from Vedanta Biosciences, Inc.