Discovery and characterization of ribosomally synthesized and post-translationally modified peptide natural products

Bacterial secondary metabolites have historically been a rich source of antibiotic compounds. The genomic era has shown that bacteria harbor the potential to produce even more natural products that could be developed into new antibiotic agents to combat the alarming rise in antibiotic- resistant pathogens. Ribosomally synthesized and post-translationally modified peptide (RiPP) natural products are genetically encoded peptide-derived secondary metabolites whose structures can be predicted based on gene sequence and identity of tailoring enzymes within the biosynthetic gene cluster. Post-translational modifications installed by the tailoring enzymes in a site-selective manner endow the compounds with structural rigidity and, in many cases, interesting biological activities. Once genome mining has been used to select RiPPs gene clusters, chemical and microbiological techniques are employed to characterize the structure, biosynthesis, antibiotic activity, and mode of action of newly discovered RiPPs. Plantazolicin (PZN) is a RiPP natural product with a rigid, polyheterocyclic structure and a remarkably species-specific antibiotic activity, targeting the pathogen Bacillus anthracis. While the highly modified natural product bears the characteristic heterocycles of a linear azol(in)e- containing RiPP, N-terminal dimethylation after cleavage of the leader peptide is also essential for bioactivity. The methyltransferase responsible for this post-translational modification is highly evolved for PZN-like substrates, with very little tolerance to mutant sequences. The underlying cause of the ultra-narrow spectrum bioactivity was investigated using a broad range of chemical and microbiological techniques. PZN was not found to have a proteinaceous target; instead, the bioactivity was found to be dependent on the diphosphatidylglycerol lipid cardiolipin. PZN is thought to localize to weakened sections of the B. anthracis membrane that contain aggregates of cardiolipin. These lipid aggregates create regions of membrane instability in B. anthracis, and PZN is able to insert into these regions, causing membrane depolarization and eventual cell lysis. Exogenous cardiolipin synergizes with PZN to enhance antibiotic potency. The citrulassin sub-family of lasso peptides contain a citrulline post-translational modification which inspired reactivity-based screening for additional citrullinated natural products from bacteria. While ubiquitous in eukaryotes, deamination in bacteria is poorly characterized and exceedingly rare. Surprisingly, the peptidyl arginine deiminase responsible for the modification of arginine to citrulline in bacteria is not known. Comparative genomics was employed to find a putative PAD enzyme, which was subsequently heterologously expressed and tested for arginine deiminase activity. The scope of citrulline post-translational modifications in the citrulassins was further investigated, and the utility of the probe to label citrulline-containing natural products in complex biological extracts was studied. The bacterial arginine deiminase responsible for the modification of peptidic substrates was investigated. The citrulline-specific reaction is useful for labeling of not just RiPPs, but other citrulline-containing natural products as well