Synthesis of a peptide derivative of microcin J25 and evaluation of antibacterial and biological activities

Document Type : Research article


1 Department of Microbiology, Science and Research Branch, Islamic Azad University, Tehran, Iran

2 Radiation Application Research School, Nuclear Science and Technology Research Institute (NSTRI), P.O.Box: 14395-836, Tehran, Iran

3 Department of Microbiology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran

4 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran.


Microcin J25 (MccJ25) is a small ribosomally synthesized antimicrobial peptide that is produced by Enterobacteriacea family especially E.coli. The present study focuses on preparation and evaluation of in vitro antimicrobial and biological properties of a new peptide derived from MccJ25. We prepared a MccJ25-derived peptide containing 14 amino acids and a single intra-molecular disulfide bond according to solid phase synthesis strategy. The purified peptide was characterized by Liquid chromatography-mass spectrometry (LC-MS) and Fourier Transform Infrared (FTIR) spectroscopy. 96-well microdilution plate assay was exerted for determination of minimum inhibitory concentration (MIC) of peptide against different bacterial strains. Cytotoxicity of the peptide derivative on HT-29 cell line assayed using MTT test. The final peptide successfully was prepared with purity more than 99.8% as determined by analytical HPLC. The evaluation of antibacterial activity of the peptide against Gram-positive and Gram- negative bacteria revealed that the peptide was very effective against E.coli 35218 with minimum inhibitory concentration (MIC) at dose 3.9 µM. The hemolytic activity toward human erythrocytes was very minimal below 0.3 %. The cell viability percentage of HT-29 cell line after 24 hours of contact with the peptide was more than 83%. The high sensitivity of E.coli strain to this new peptide derived from MccJ25 and through minimal toxicity to cancerous cell, suggesting that above synthesized peptide could be considered as a bioactive compound for further investigations.


Main Subjects

Jensen H, Hamill P and Hancock REW. Peptide
antimicrobial agents.
Clin. Microbiol. Rev. (2006) 19:

Hancock REW and Chapple DS. Peptide antibiotics.

Antimicrob. Agents Chemother.
(1999) 43: 1317-23.
Sang Y and Blecha F. Antimicrobial peptides and

bacteriocins: alternatives to traditional antibiotics.

Anim. Health Res. Rev.
(2008) 9: 227-35.
Yang SC, Lin CH, Sung CT and Fang JY.

Antimicrobial of bacterocins: application of foods and

Front. Microbiol. (2014) 5: 241-7.
Snyder AB and Worobo RW. Chemical and genetic

characterization of bacteriocins: antimicrobial peptides

for food safety.
J. Sci. Food Agric. (2014) 94: 28-44.
Seo MD, Won HS, Kim JH, Mishig-Ochir T and Lee BJ.

Anti-microbial peptides for therapeutic applications: a

Molecules (2012) 17: 12276-86.
Budic M, Rijavec M, Petkovsek Z and Zgur-Bertok D.







Synthesis of a Peptide Derivative of MicrocinJ251275
Escherichia coli
bacteriocins: antimicrobial efficacy
and prevalence among isolates from patients with

PLoS One (2012) 6: e28769.
Kumar Tiwari S, Sutyak Noll K, Cavera VL and

Chikindas ML. Improved antimicrobial activities of

synthetic-hybrid bacteriocins designed from enterocin

E50-52 and pediocin PA-1.
Appl. Environ. Microbiol.
(2015) 81: 1661-7.

Hassan M, Kjos IF, Nes DB and Lotfipour M. Natural

antimicrobial peptides from bacteria: characteristics

and potential applications to fight against antibiotic

J. Appl. Microbiol. (2015) 113: 723-36.
Chalon MC, Bellomio A, Solbiati JO, Morero RD,

Farias RN and Vincent PA. Tyrosine
9 is the key amino
acid in microcinJ25 superoxide over production.

FEMS Microbiol. Lett.
(2009) 300: 90-6.
Vincent PA and Delgado MA. Farias RN and Salomon

RA. Inhibition of
Salmonella enterica serovars by
microcin J25.
FEMS Microbiol. Lett. (2004) 236:

Rintoul MR, De Arcuri BF, Salomon RA, Farias RN

and Morero RD. The antibacterial action of microcin

J25: evidence for disruption of cytoplasmic membrane

energization in
Salmonella Newport. FEMS Microbiol.
(2001) 204: 265-70.
Jia Pan S, Cheung WL, Fung HK, Floudas CA and

Link AJ. Computational design of the lasso peptide

antibiotic microcin J25.
Protein Eng. Des. Sel. (2011)
24: 275-82.

Rebuffat S. Microcins in action: amazing defense

strategies of
Enterobacteria. Biochem. Soc. Trans.
(2012) 40: 1456-62.

Dupuy F and Morero R. Microcin J25 membrane

interaction: Selectivity toward gel phase.
Biophys. Acta.
(2011) 1808: 1764-71.
Hegemann JD, Zimmermann M, Xie X and Marahiel

MA. Lasso peptides: an intriguing class of bacterial

natural products.
Acc. Chem. Res. (2015) 48: 1909-19.
Adelman K, Yuzenkova, J, La Porta A, Zenkin N, Lee

J, Lis JT, Borukhov S, Wang MD and Severinov K.

Molecular mechanism of transcription inhibition by

peptide antibiotic microcin J25.
Mol. Cell (2004) 14:

Jia Pan S and James Link A. Sequence diversity in

the lasso peptide framework: discovery of functional

microcin J25 variants with multiple amino acid

J. Am. Chem. Soc. (2011) 133: 5016-23.
Kuznedelov K, Semenova E, Knappe TA,

Mukhamedjarov D, Srivastava A, Chatterjee S, Ebright

RH, Marahiel M and Severinov K. The antibacterial

threaded-lasso peptide capistruin inhibits bacterial

RNA polymerase.
J. Mol. Biol. (2011) 412: 842-8.
Pavlova O, Mukhopadhyay J, Sineva E, Ebright

RH and Severinov K. Systematic structure-activity

analysis of microcin J25.
J. Biol. Chem. (2008) 283:

Destoumieux-Garzon D, Duquesne S, Peduzzi J,

Goulard C, Desmadril M, Letellier L, Rebuffat S and

Boulanger P. The iron–siderophore transporter FhuA

is the receptor for the antimicrobial peptide microcin

J25: role of the microcin Val11–Pro16 β-hairpin region

in the recognition mechanism.
Biochem. J. (2005) 389:

Chiuchiolo M, Delgado M, Farias R and Salomon

R. Growth-phase dependent expression of the

cyclopeptide antibiotic microcin J25.
J. Bacteriol.
(2005) 183: 1755-64.

Severinov K, Semenova E, Kazakov A, Kazakov

T and Gelfand MS. Low-molecular-weight post-

translationally modified microcins.
Mol. Microbiol.
(2007) 65: 1380-94.

Bellomio A, Vincent PA, Arcuri BF, Farı ́as RN and

Morero RD. Microcin J25 has dual and independent

mechanisms of action in Escherichia coli: RNA

polymerase inhibition and increased superoxide

J. Bacteriol. (2007) 189: 4180-6.
Soudy R, Wang L and Kaur K. Synthetic peptides

derived from the sequence of a lasso peptide microcin

J25 show antibacterial activity.
Bioorg. Med. Chem.
(2012) 20: 1794-800.

Hammami R, Bedard F, Gomma A, Subirade M, Biron

E and Fliss I. Lasso-inspired peptides with distinct

antibacterial mechanisms.
Amino Acids (2015) 47:

Ferguson AL, Zhang S, Dikiy I, Panagiotopoulos AZ,

Debenedetti PG and James Link A. An experimental

and computational investigation of spontaneous lasso

formation in microcin J25.
Biophys. J. (2010) 99:

Wilson KA, Kalkum M, Ottesen J,
Yuzenkova J,
Chait BT, Landick R, Muir T, Severinov K
and Darst
SA. Structure of microcin J25, a peptide inhibitor
bacterial RNA polymerase, is a lassoed tail. J. Am
Chem. Soc. (2003) 125: 12475-83.

Rosengren KJ, Clark
RJ, Daly NL, Goransson U,
Jones A and Craik
DJ. Microcin J25 has a threaded
sidechain-to-backbone ring
structure and not a head-
to-tail cyclized backbone. J.
Am. Chem. Soc. (2003)
125: 12464-74.

Atherton E and Sheppard R. Fluorenylmethoxycarbonyl-

polyamide solid phase peptide synthesis general

principles and development. Solid phase peptide

synthesis. A practical approach, Oxford Information

Press. (1989) 25-38.

Balouiri M, Sadiki M and KoraichiIbnsoud S. Methods

in-vitro evaluating antimicrobial activity: a review.
J. Pharm. Anal.
(2016) 6: 71-9.
Semenova E, Yuzenkova Y, Peduzzi J, Rebuffat S and

Severinov K. Structure-activity analysis of microcin

J25: distinct parts of the threaded lasso molecule

are responsible for interaction with bacterial RNA

J. Bacteriol. (2005) 187: 3859-63.
Bellomio A, Vincent PA, de Arcuri BF, Salomon RA,

Morero RD and Farias RN. The microcin J25 beta-

hairpin region is important for antibiotic uptake but

not for RNA polymerase and respiration inhibition.

Biochem. Biophys. Res. Commun.
(2004) 325: 1454-8.
Mukhopadhyay J, Sineva E, Knight J, Levy RM and



























Mazaheri Tehrania M et al. / IJPR (2019), 18 (3): 1264-12761276
Ebright RH. Antibacterial peptide microcin J25 inhibits

transcription by binding within and obstructing the

RNA polymerase secondary channel.
Mol. Cell (2004)
14: 739-51.

Adelman K, Yuzenkova J, La Porta A, Zenkin N, Lee

J, Lis JT, Borukhov S, Wang MD and Severinov K.

Molecular mechanism of transcription inhibition by

peptide antibiotic Microcin J25.
Mol. Cell (2004) 14:

Miller JH. A Short Course in Bacterial Genetics. Cold

Spring Harbor Laboratory, Cold Spring Harbor, NY


Mukhopadhyay J, Kapanidis AN, Mekler V,

Kortkhonjia E, Ebright YW and Ebright RH.

Translocation of sigma (70) with RNA polymerase

during transcription: fluorescence resonance energy

transfer assay for movement relative to DNA.
106: 453-63.



Schlageck JG, Baughman M and Yarbrough LR.

Spectroscopic techniques for study of phosphodiester

bond formation by Escherichia coli RNA polymerase.

J. Biol. Chem.
(1979) 254: 12074-7.
Whitcomb DC and Lowe ME. Human pancreatic

digestive enzymes.
Dig. Dis. Sci. (2007) 52: 1-17.
Gray GM and Cooper HL. Protein digestion and

Gastroenterology. (1971) 61: 535-44.
Mohamed MF, Abdelkhalek A and Seleem MN.

Evaluation of short synthetic antimicrobial peptides

for treatment of drug-resistant and intracellular

Staphylococcus aureus
. Sci. Rep. (2016) 6: 29707.
Soudy R, Etayash H, Bahadorani K, Lavasanifar A and

Kaur K. Breast cancer targeting peptide binds keratin

1: a new molecular marker for targeted drug delivery

to breast cancer.
Mol. Pharm. (2017) 14: 593-604.