Plantazolicin (PZN) is a ribosomally synthesized and post-translationally modified natural product from Bacillus methylotrophicus FZB42 and Bacillus pumilus. Extensive tailoring to 12 of the 14 amino acid residues in the mature natural product endows PZN with not only a rigid, polyheterocyclic structure, but also antibacterial activity. Here we report the remarkably discriminatory activity of PZN toward Bacillus anthracis, which rivals a previously described gamma (γ) phage lysis assay in distinguishing B. anthracis from other members of the Bacillus cereus group. We evaluate the underlying cause of this selective activity by measuring the RNA expression profile of PZN-treated B. anthracis, which revealed significant up-regulation of genes within the cell envelope stress response. PZN depolarizes the B. anthracis membrane like other cell envelope-acting compounds but uniquely localizes to distinct foci within the envelope. Selection and whole-genome sequencing of PZN-resistant mutants of B. anthracis implicate a relationship between the action of PZN and cardiolipin (CL) within the membrane. Exogenous CL increases the potency of PZN in wild type B. anthracis and promotes the incorporation of fluorescently tagged PZN in the cell envelope. We propose that PZN localizes to and exacerbates structurally compromised regions of the bacterial membrane, which ultimately results in cell lysis.Keywords: anthrax; antibiotic; Bacillus anthracis; membrane depolarization; mode of action; oxazole; pathogen specific antibiotic; ribosomally synthesized and post-translationally modified natural product; thiazole

Peptide antibiotics represent a class of conformationally constrained natural products of growing pharmaceutical interest. Plantazolicin (PZN) is a linear, polyheterocyclic natural product with highly selective and potent activity against the anthrax-causing bacterium, Bacillus anthracis. The bioactivity of PZN is contingent on dimethylation of its N-terminal Arg residue by an S-adenosylmethionine-dependent methyltransferase. Here, we explore the substrate tolerances of two homologous PZN methyltransferases by carrying out kinetic analyses of the enzymes against a synthetic panel of truncated PZN analogs containing the N-terminal Arg residue. X-ray cocrystal structures of the PZN methyltransferases with each of these heterocycle-containing substrates provide a rationale for understanding the strict substrate specificity of these enzymes. Kinetic studies of structure-guided, site-specific variants allowed for the assignment of residues governing catalysis and substrate scope. Microbiological testing further revealed that upon dimethylation of the N-terminal Arg, a pentaheterocyclized PZN analog retained potent anti-B. anthracis activity, nearly equal to that of full-length PZN. These studies may be useful in the biosynthetic engineering of natural product analogs with different bioactivity profiles, as demonstrated by our identification of a truncated plantazolicin derivative that is active against methicillin-resistant Staphylococcus aureus (MRSA).

Peptide antibiotics represent a class of conformationally constrained natural products of growing pharmaceutical interest. Plantazolicin (PZN) is a linear, polyheterocyclic natural product with highly selective and potent activity against the anthrax-causing bacterium, Bacillus anthracis. The bioactivity of PZN is contingent on dimethylation of its N-terminal Arg residue by an S-adenosylmethionine-dependent methyltransferase. Here, we explore the substrate tolerances of two homologous PZN methyltransferases by carrying out kinetic analyses of the enzymes against a synthetic panel of truncated PZN analogs containing the N-terminal Arg residue. X-ray cocrystal structures of the PZN methyltransferases with each of these heterocycle-containing substrates provide a rationale for understanding the strict substrate specificity of these enzymes. Kinetic studies of structure-guided, site-specific variants allowed for the assignment of residues governing catalysis and substrate scope. Microbiological testing further revealed that upon dimethylation of the N-terminal Arg, a pentaheterocyclized PZN analog retained potent anti-B. anthracis activity, nearly equal to that of full-length PZN. These studies may be useful in the biosynthetic engineering of natural product analogs with different bioactivity profiles, as demonstrated by our identification of a truncated plantazolicin derivative that is active against methicillin-resistant Staphylococcus aureus (MRSA).