Paenitracins, a novel family of bacitracin-type nonribosomal peptide antibiotics produced by plant-associated <i>Paenibacillus</i> species

The growing threat of antimicrobial resistance necessitates the discovery of novel antibiotics with activity against drug-resistant pathogens. Members of the genus <i>Paenibacillus</i> are a rich source of nonribosomal peptides (NRPs), including well-known antibiotics such as polymyxins, paenibacterin, and tridecaptins. Here, we use a targeted mass spectrometry query language (MassQL)-based approach to identify the NRPs produced by a collection of 227 taxonomically diverse plant-associated <i>Paenibacillus</i> strains, providing detailed insights into their NRP-producing potential. Using MassQL to zoom in specifically on NRPs containing basic amino acids, we discovered a novel family of bacitracins, which we designated paenitracins. The paenitracins are the first bacitracin-type peptides reported in <i>Paenibacillus</i> and are distinguished from canonical bacitracins by three previously unseen amino acid substitutions. The paenitracins exhibit potent activity against gram-positive pathogens, including vancomycin-resistant <i>Enterococcus faecium</i> E155. Our work provides a novel metabolomics-guided and genomics-guided workflow for the discovery of bioactive NRPs as a strategy to prioritize natural product chemical space and accelerate antibiotic discovery.IMPORTANCEMembers of the genus <i>Paenibacillus</i> play an important role in soil ecology, producing a range of important nonribosomal peptides (NRPs). A collection of plant-associated <i>Paenibacillus</i> spp. were analyzed for their phylogenetic and metabolic diversity. We developed a novel discovery pipeline that combines feature-based molecular networking with mass spectrometry query language queries to systematically prioritize bioactive NRPs containing basic amino acids. Thus, we provide a comprehensive genus-wide inventory of NRPs produced by <i>Paenibacillus</i> spp. We thereby identified the paenitracins, a new sub-family of bacitracins active against multidrug-resistant gram-positive pathogens. Our pipeline enables the discovery of novel peptidic natural products to accelerate the prioritization of chemical space for antibiotics.