The Product of Yersinia pseudotuberculosis mcc Operon Is a Peptide-Cytidine Antibiotic Activated Inside Producing Cells by the TldD/E Protease.

Microcin C is a heptapeptide-adenylate antibiotic produced by some strains of Escherichia coli. Its peptide part is responsible for facilitated transport inside sensitive cells where it is proteolyzed with release of a toxic warhead-a nonhydrolyzable aspartamidyl-adenylate, which inhibits aspartyl-tRNA synthetase. Recently, a microcin C homologue from Bacillus amyloliquefaciens containing a longer peptide part modified with carboxymethyl-cytosine instead of adenosine was described, but no biological activity of this compound was revealed. Here, we characterize modified peptide-cytidylate from Yersinia pseudotuberculosis. As reported for B. amyloliquefaciens homologue, the initially synthesized compound contains a long peptide that is biologically inactive. This compound is subjected to endoproteolytic processing inside producing cells by the evolutionary conserved TldD/E protease. As a result, an 11-amino acid long peptide with C-terminal modified cytosine residue is produced. This compound is exported outside the producing cell and is bioactive, inhibiting sensitive cells in the same way as E. coli microcin C. Proteolytic processing inside producing cells is a novel strategy of peptide-nucleotide antibiotics biosynthesis that may help control production levels and avoid toxicity to the producer.

A Trojan-Horse Peptide-Carboxymethyl-Cytidine Antibiotic from Bacillus amyloliquefaciens.

Microcin C and related antibiotics are Trojan-horse peptide-adenylates. The peptide part is responsible for facilitated transport inside the sensitive cell, where it gets processed to release a toxic warhead-a nonhydrolyzable aspartyl-adenylate, which inhibits aspartyl-tRNA synthetase. Adenylation of peptide precursors is carried out by MccB THIF-type NAD/FAD adenylyltransferases. Here, we describe a novel microcin C-like compound from Bacillus amyloliquefaciens. The B. amyloliquefaciens MccB demonstrates an unprecedented ability to attach a terminal cytidine monophosphate to cognate precursor peptide in cellular and cell free systems. The cytosine moiety undergoes an additional modification-carboxymethylation-that is carried out by the C-terminal domain of MccB and the MccS enzyme that produces carboxy-SAM, which serves as a donor of the carboxymethyl group. We show that microcin C-like compounds carrying terminal cytosines are biologically active and target aspartyl-tRNA synthetase, and that the carboxymethyl group prevents resistance that can occur due to modification of the warhead. The results expand the repertoire of known enzymatic modifications of peptides that can be used to obtain new biological activities while avoiding or limiting bacterial resistance.