RESUMEN
The Escherichia coli microcin C (McC) and related compounds are potent Trojan horse peptide-nucleotide antibiotics. The peptide part facilitates transport into sensitive cells. Inside the cell, the peptide part is degraded by nonspecific peptidases releasing an aspartamide-adenylate containing a phosphoramide bond. This nonhydrolyzable compound inhibits aspartyl-tRNA synthetase. In addition to the efficient export of McC outside the producing cells, special mechanisms have evolved to avoid self-toxicity caused by the degradation of the peptide part inside the producers. Here, we report that histidine-triad (HIT) hydrolases encoded in biosynthetic clusters of some McC homologs or by standalone genes confer resistance to McC-like compounds by hydrolyzing the phosphoramide bond in toxic aspartamide-adenosine, rendering them inactive.IMPORTANCE Uncovering the mechanisms of resistance is a required step for countering the looming antibiotic resistance crisis. In this communication, we show how universally conserved histidine-triad hydrolases provide resistance to microcin C, a potent inhibitor of bacterial protein synthesis.
Asunto(s)
Antibacterianos/metabolismo , Bacteriocinas/genética , Hidrolasas/metabolismo , Familia de Multigenes , Myxococcales/enzimología , Myxococcales/genética , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Vías Biosintéticas , Farmacorresistencia Bacteriana , Escherichia coli/genética , Hidrolasas/genética , Myxococcales/efectos de los fármacos , Operón , Péptidos/metabolismo , Péptidos/farmacologíaRESUMEN
Microcin C (McC) is a peptide adenylate antibiotic produced by Escherichiacoli cells bearing a plasmid-borne mcc gene cluster. Most MccA precursors, encoded by validated mcc operons from diverse bacteria, are 7 amino acids long, but the significance of this precursor length conservation has remained unclear. Here, we created derivatives of E. colimcc operons encoding longer precursors and studied their synthesis and bioactivities. We found that increasing the precursor length to 11 amino acids and beyond strongly decreased antibiotic production. We found this decrease to depend on several parameters. First, reiterative synthesis of the MccA peptide by the ribosome was decreased at longer mccA open reading frames, leading to less efficient competition with other messenger RNAs. Second, the presence of a formyl group at the N-terminal methionine of the heptameric peptide had a strong stimulatory effect on adenylation by the MccB enzyme. No such formyl group stimulation was observed for longer peptides. Finally, the presence of the N-terminal formyl on the heptapeptide adenylate stimulated bioactivity, most likely at the uptake stage. Together, these factors should contribute to optimal activity of McC-like compounds as 7-amino-acid peptide moieties and suggest convergent evolution of several steps of the antibiotic biosynthesis pathway and their adjustment to sensitive cell uptake machinery to create a potent drug.IMPORTANCEEscherichia coli microcin C (McC) is a representative member of peptide-nucleotide antibiotics produced by diverse microorganisms. The vast majority of biosynthetic gene clusters responsible for McC-like compound production encode 7-amino-acid-long precursor peptides, which are C-terminally modified by dedicated biosynthetic enzymes with a nucleotide moiety to produce a bioactive compound. In contrast, the sequences of McC-like compound precursor peptides are not conserved. Here, we studied the consequences of E. coli McC precursor peptide length increase on antibiotic production and activity. We show that increasing the precursor peptide length strongly decreases McC production by affecting multiple biosynthetic steps, suggesting that the McC biosynthesis system has evolved under significant functional constraints to maintain the precursor peptide length.
Asunto(s)
Antibacterianos/metabolismo , Antibacterianos/farmacología , Bacteriocinas/metabolismo , Bacteriocinas/farmacología , Escherichia coli/metabolismo , Biosíntesis de Proteínas , Ribosomas/metabolismo , Bacteriocinas/genética , Análisis Mutacional de ADN , Escherichia coli/genética , N-Formilmetionina/metabolismo , Sistemas de Lectura Abierta , PlásmidosRESUMEN
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.
Asunto(s)
Antibacterianos/biosíntesis , Proteínas Bacterianas/metabolismo , Operón/genética , Péptido Hidrolasas/metabolismo , Yersinia pseudotuberculosis/metabolismo , Antibacterianos/química , Citidina/biosíntesis , Citidina/química , Citidina/genética , Péptidos/química , Péptidos/genética , Péptidos/metabolismo , Yersinia pseudotuberculosis/citología , Yersinia pseudotuberculosis/genéticaRESUMEN
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.
Asunto(s)
Antibacterianos/farmacología , Aspartato-ARNt Ligasa/antagonistas & inhibidores , Bacillus amyloliquefaciens/química , Bacteriocinas/farmacología , Inhibidores Enzimáticos/farmacología , Antibacterianos/química , Antibacterianos/aislamiento & purificación , Aspartato-ARNt Ligasa/genética , Aspartato-ARNt Ligasa/metabolismo , Bacteriocinas/química , Bacteriocinas/aislamiento & purificación , Biología Computacional , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/aislamiento & purificación , Estructura MolecularRESUMEN
UNLABELLED: Escherichia coli microcin C (McC) consists of a ribosomally synthesized heptapeptide attached to a modified adenosine. McC is actively taken up by sensitive Escherichia coli strains through the YejABEF transporter. Inside the cell, McC is processed by aminopeptidases, which release nonhydrolyzable aminoacyl adenylate, an inhibitor of aspartyl-tRNA synthetase. McC is synthesized by the MccB enzyme, which terminally adenylates the MccA heptapeptide precursor MRTGNAN. Earlier, McC analogs with shortened peptide lengths were prepared by total chemical synthesis and were shown to have strongly reduced biological activity due to decreased uptake. Variants with longer peptides were difficult to synthesize, however. Here, we used recombinant MccB to prepare and characterize McC-like molecules with altered peptide moieties, including extended peptide lengths. We find that N-terminal extensions of E. coli MccA heptapeptide do not affect MccB-catalyzed adenylation and that some extended-peptide-length McC analogs show improved biological activity. When the peptide length reaches 20 amino acids, both YejABEF and SbmA can perform facilitated transport of toxic peptide adenylates inside the cell. A C-terminal fusion of the carrier maltose-binding protein (MBP) with the MccA peptide is also recognized by MccB in vivo and in vitro, allowing highly specific adenylation and/or radioactive labeling of cellular proteins. IMPORTANCE: Enzymatic adenylation of chemically synthesized peptides allowed us to generate biologically active derivatives of the peptide-nucleotide antibiotic microcin C with improved bioactivity and altered entry routes into target cells, opening the way for development of various McC-based antibacterial compounds not found in nature.