Reiterative synthesis by the ribosome and recognition of the N-terminal formyl group by biosynthetic machinery contribute to evolutionary conservation of the length of antibiotic microcin c peptide precursor

Inna Zukher, Michael Pavlov, Darya Tsibulskaya, Alexey Kulikovsky, Tatyana Zyubko, Dmitry Bikmetov, Marina Serebryakova, Satish K. Nair, Måns Ehrenberg, Svetlana Dubiley, Konstantin Severinov

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

Microcin C (McC) is a peptide adenylate antibiotic produced by Escherichia coli 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. coli mcc 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. IMPORTANCE Escherichia 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.

Original languageEnglish (US)
Article numbere00768-19
JournalmBio
Volume10
Issue number2
DOIs
StatePublished - Mar 1 2019

Fingerprint

Ribosomes
Anti-Bacterial Agents
Peptides
Amino Acids
Operon
Multigene Family
Escherichia coli
Nucleotides
microcin
Enzymes
Methionine
Open Reading Frames
Plasmids
Bacteria
Messenger RNA

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Virology

Keywords

  • Antibiotic
  • Microcin
  • Ribosome
  • Translation initiation

Cite this

Zukher, Inna ; Pavlov, Michael ; Tsibulskaya, Darya ; Kulikovsky, Alexey ; Zyubko, Tatyana ; Bikmetov, Dmitry ; Serebryakova, Marina ; Nair, Satish K. ; Ehrenberg, Måns ; Dubiley, Svetlana ; Severinov, Konstantin. / Reiterative synthesis by the ribosome and recognition of the N-terminal formyl group by biosynthetic machinery contribute to evolutionary conservation of the length of antibiotic microcin c peptide precursor. In: mBio. 2019 ; Vol. 10, No. 2.
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abstract = "Microcin C (McC) is a peptide adenylate antibiotic produced by Escherichia coli 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. coli mcc 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. IMPORTANCE Escherichia 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.",
keywords = "Antibiotic, Microcin, Ribosome, Translation initiation",
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Reiterative synthesis by the ribosome and recognition of the N-terminal formyl group by biosynthetic machinery contribute to evolutionary conservation of the length of antibiotic microcin c peptide precursor. / Zukher, Inna; Pavlov, Michael; Tsibulskaya, Darya; Kulikovsky, Alexey; Zyubko, Tatyana; Bikmetov, Dmitry; Serebryakova, Marina; Nair, Satish K.; Ehrenberg, Måns; Dubiley, Svetlana; Severinov, Konstantin.

In: mBio, Vol. 10, No. 2, e00768-19, 01.03.2019.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Reiterative synthesis by the ribosome and recognition of the N-terminal formyl group by biosynthetic machinery contribute to evolutionary conservation of the length of antibiotic microcin c peptide precursor

AU - Zukher, Inna

AU - Pavlov, Michael

AU - Tsibulskaya, Darya

AU - Kulikovsky, Alexey

AU - Zyubko, Tatyana

AU - Bikmetov, Dmitry

AU - Serebryakova, Marina

AU - Nair, Satish K.

AU - Ehrenberg, Måns

AU - Dubiley, Svetlana

AU - Severinov, Konstantin

PY - 2019/3/1

Y1 - 2019/3/1

N2 - Microcin C (McC) is a peptide adenylate antibiotic produced by Escherichia coli 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. coli mcc 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. IMPORTANCE Escherichia 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.

AB - Microcin C (McC) is a peptide adenylate antibiotic produced by Escherichia coli 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. coli mcc 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. IMPORTANCE Escherichia 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.

KW - Antibiotic

KW - Microcin

KW - Ribosome

KW - Translation initiation

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