SignificanceMany bacteria produce antimicrobial peptides for survival under stressful conditions. Some of these antimicrobial peptides are lasso peptides, which have a unique lasso-like topology and have generated great interest as a result of their stability in harsh conditions and amenability to functional engineering. In this study, we determined crystal structures of two lasso peptides, microcin J25 and capistruin, bound to their natural enzymatic target, the bacterial RNA polymerase (RNAP). The structures define peptide inhibitor–RNAP interactions that are important for inhibition and provide detailed insight into how the peptides inhibit RNAP function. This work provides a structural basis to guide the design of more potent lasso peptide antimicrobial approaches.

Highlights d Metabolite library from human microbiota screened for direct agonism of 241 GPCRs d Taxa-specific primary metabolites agonize individual GPCRs or broad GPCR families d Bacteria agonize receptors linked to metabolism, neurotransmission, and immunity d Simple bacterial metabolites may play a role in modulating host pathways

Using the amber suppression approach, four noncanonical amino acids (ncAAs) were used to replace existing amino acids at four positions in lasso peptide microcin J25 (MccJ25). The lasso peptide biosynthesis enzymes tolerated all four ncAAs and produced antibiotics with efficacy equivalent to wild-type in some cases. Given the rapid expansion of the genetically encoded ncAA pool, this study is the first to demonstrate an expedient method to significantly increase the chemical diversity of lasso peptides.

Toxin–antitoxin (TA) systems interfere with essential cellular processes and are implicated in bacterial lifestyle adaptations such as persistence and the biofilm formation. Here, we present structural, biochemical, and functional data on an uncharacterized TA system, the COG5654–COG5642 pair. Bioinformatic analysis showed that this TA pair is found in 2,942 of the 16,286 distinct bacterial species in the RefSeq database. We solved a structure of the toxin bound to a fragment of the antitoxin to 1.50 Å. This structure suggested that the toxin is a mono-ADP-ribosyltransferase (mART). The toxin specifically modifies phosphoribosyl pyrophosphate synthetase (Prs), an essential enzyme in nucleotide biosynthesis conserved in all organisms. We propose renaming the toxin ParT for Prs ADP-ribosylating toxin and ParS for the cognate antitoxin. ParT is a unique example of an intracellular protein mART in bacteria and is the smallest known mART. This work demonstrates that TA systems can induce bacteriostasis through interference with nucleotide biosynthesis.

The continued prevalence of COVID-19 and the emergence of new variants has once again put the spotlight on the need for the identification of SARS-CoV-2 antivirals. The human microbiome produces an array of small molecules with bioactivities (e.g., host receptor ligands), but its ability to produce antiviral small molecules is relatively underexplored.