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

The growing threat of antimicrobial resistance necessitates the discovery of novel antibiotics with activity against drug-resistant pathogens. Members of the genus Paenibacillus are a rich source of nonribosomal peptides (NRPs), including well-known antibiotics such as polymyxins, paenibacterin and tridecaptins. Here we use a targeted Mass-QL-based mass spectrometry approach to identify the NRPs produced by a collection of 227 taxonomically diverse plant-associated Paenibacillus 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 Paenibacillus, 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 Enterococcus faecium E155. Our work provides a novel metabolomics- 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 Paenibacillus play an important role in soil ecology, producing a range of important nonribosomal peptides (NRPs) that protect their eukaryotic host. A collection of plant-associated Paenibacillus spp. was analyzed for their phylogenetic and metabolic diversity. We developed a novel discovery pipeline that combines feature-based molecular networking with MassQL queries to systematically prioritize bioactive NRPs containing basic amino acids. Thus we provide a comprehensive genus-wide inventory of NRPs produced by Paenibacillus spp. We thereby identified the paenitracins, a new subfamily 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.