Part:BBa_K1931014
T7-RBS-Microcin E492-MBP-Terminator
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Sequence and Features
This part codes for the bacteriocin Microcin E492 with MBP fusion protein
Ionis_Paris 2020 Documentation
Group: Ionis_Paris 2020
Author: Giulia Criseo
Summary: Team Ionis_Paris 2020 contributed to this part by adding information learnt from the literature, a 3D structure of the part and existing experiments to characterize the part. The original composite part was not correctly annotated, so we performed a blast to add the features of each sequence composing the part. We did not have the time to test this part in the lab but it could be very useful in the case of our project, BacTail which goal is to fight against antimicrobial resistance with a antimicrobial peptide producing bacterium visit our wiki.
Usage
Bacteriocins are antimicrobial proteins naturally synthesized by bacteria to inhibit the growth of closely related species[1]. They are produced by the strains Eubacteria and Archaebacteria and are excreted in order to specifically recognize and destroy certain bacteria[2]. Bacteriocins can kill using different mechanisms, such as the formation of membrane pores. They are widely used as preservatives in the food industry or as antibiotics in the health industry[3].
Adding part features
To determine the full annotation of the part sequence, we first tried to find the corresponding sequence of the Microcin E492 and MBP on NCBI and Uniprot. No sequences matched so we did several Blasts between this sequence and other sequences that team iGEM16_Cornell_NY used. We first determined the terminator part and then the MBP sequence which seem to be common throughout their different parts. We then estimated the Microcin E492 sequence. We added the following features to the part: Microcin E492, TEV site, 6X His Tag, MBP and Terminator.
We were unable to access the modification of part features in the Registry. This is how the part should be annotated: T7 Promoter (1-46), RBS (47-80), Microcin E492 (81-386), 6X-His tag (453-470), TEV site (387-407), MBP (408-1591), Terminator (409, 1630).
Biology
Microcin E492 (Mcc E492) is a type of bacteriocin that is active against ‘’Erwinia’’, Salmonella, Escherichia coli, Enterobacter, Citrobacter and Klebsiella [4]. MceA, its structural gene, was cloned and sequenced that codes for a precursor of 99 or 103 amino acids. In order to achieve its antibacterial activity, Mcc E492 creates pores in the target’s membrane, which induces membrane depolarization [1]. Until now, Mcc E492 is the only channel-forming microcin that has been described and represents a model of low-molecular-weight pore-forming bacteriocins [5].
Bacterial strains, Culture conditions and Microcin detection
Mcc E492 was produced by a recombinant E. coli strain; VCS 257 (AmpR). M63 minimal medium was used for the following experiments. The media for petri dishes were solidified using 12g or 6g of E-type agar per liter. Bacterial cultures were propagated in BHI at 37°C [5].
Purification of the microcin
Microcin E492 was prepurified by solid-phase extraction and purified by reverse-phase high-pressure liquid chromatography (RP-HPLC) [6].
Analysis of the microcin
A Biflex matrix-assisted laser desorption ionization-time of flight-mass spectrometer (MALDI-TOF-MS) was used to determine the molecular mass of the microcin. Ionization was performed with a 337nm nitrogen laser beam. Spectra were obtained in the positive ionization mode. Electrospray ionization mass spectrometry (ESMS) analysis was accomplished on a VG BioQ mass spectrometer. MALDI-TOF-MS analysis showed a unique peak at 7,887.8 Da. Thanks to ESMS, they determined the accurate molecular mass of Microcin E492 to 7,886.68 ± 0.52 Da [6].
Mcc E492 was digested with endoproteinase Asp-N and the resulting peptides were analyzed by liquid chromatography-mass spectroscopy (LC-MS) (Table 1). Microcin E492 was observed to be an 84-amino-acid polypeptide. Its sequence is GETDPNTQLL10NDLGNNMAWG20AALGAP
G*G*L+G30*SA*A*LG*AA*G*G*A40*L+QTVGQGLID50HGPVNVPIPV60 LIGPSWNGSG70SGYNSATSSS80GSGS. The asterisks indicate conserved amino acids, and the plus signs indicate semiconserved amino acids [6]
Table from : Pons, Anne-Marie, et al. "Microcin E492 is an unmodified peptide related in structure to colicin V." Antimicrobial agents and chemotherapy 46.1 (2002): 229-230.
Ability of microcin E492 to induce apoptosis
Microcin E492 is a low-molecular-mass pore-forming bacteriocin produced by Klebsiella pneumoniae. More generally, mcc E492 is active on several Enterobacteriaceae strains[4].
Microcin E492 was produced by E. coli VCS 257, extracted and purified. Several human cell lines were used for the following experiments. Cytotoxicity of microcin E492 was estimated on various human cell lines with different morphologies and physiology[1]. Cell viability after 24h incubation with mcc E492 is shown in Table 2.
Table from : Hetz, Claudio, et al. "Microcin E492, a channel-forming bacteriocin from Klebsiella pneumoniae, induces apoptosis in some human cell lines." Proceedings of the National Academy of Sciences 99.5 (2002): 2696-2701.
Microcin E492 induces apoptosis in HeLa cells
HeLa cells were selected to evaluate the cytotoxic effect of microcin E492. PI exclusion method was chosen to detect dead cells. Once, the cell loses its membrane integrity, PI enters inside the cell, binds to its DNA and the fluorescence is proportional to the amount of DNA. The results showed that apoptosis was induced in HeLa cells after treatment with mcc E4921.
High doses of Microcin E492 induce necrosis and apoptosis
Most cytotoxic agents that generate apoptosis at low concentrations, induce necrosis at higher dose [8]. Phosphatidylserine exposure at the cell surface varies in early stages of apoptosis and this can be assessed by the binding of annexin V labelled with FITC. HeLa cells treated with 512 AU/ml microcin E492 for 12h induced 32% apoptotic cells and 9% necrotic cells. When these cells are incubated with twice the dose of mcc E492 (1,024 AU/ml), the apoptotic population decreases to 25%, whereas the necrotic one reaches 32%[1] (Figure 1).
Figure from : Hetz, Claudio, et al. "Microcin E492, a channel-forming bacteriocin from Klebsiella pneumoniae, induces apoptosis in some human cell lines." Proceedings of the National Academy of Sciences 99.5 (2002): 2696-2701.
Simulating a 3D visualization of part BBa_K1931014
Due to the lack of available information, we tred to give an approximative view of the resulting fusion protein. Microcin E492 has not been characterized by crystallography and we do not have any information on the MBP (maltose binding protein) used in this part, as many different kinds exist. We tried to approximate the structure of the MicrocinE492-MBP complex. We used the crystal structure of E. coli MccB (3H9Q on PDB) and the crystal structure of ligand-free high-affinity maltose-binding protein (1N3X on PDB) to make this structure.
Conclusion
Microcin E492 is a pore-forming bacteriocin that engenders apoptosis in human cell lines. The fact that mcc E492 has a low molecular weight (7,887.8 Da) makes it interesting to study the toxin-receptor interaction and the introduction into the membrane [1].
References
[1] Hetz, Claudio, et al. "Microcin E492, a channel-forming bacteriocin from Klebsiella pneumoniae, induces apoptosis in some human cell lines." Proceedings of the National Academy of Sciences 99.5 (2002): 2696-2701.
[2] Kolter, Roberto, and Felipe Moreno. "Genetics of ribosomally synthesized peptide antibiotics." Annual review of microbiology46.1 (1992): 141-161.
[3] Jack, Ralph W., John R. Tagg, and Bibek Ray. "Bacteriocins of gram-positive bacteria." Microbiological reviews 59.2 (1995): 171-200.
[4] de Lorenzo, Víctor. "Isolation and characterization of microcin E 492 fromKlebsiella pneumoniae." Archives of microbiology 139.1 (1984): 72-75.
[5] Sable, Sophie, et al. "Antibacterial activity evaluation of microcin J25 against diarrheagenic Escherichia coli." Applied and environmental microbiology 66.10 (2000): 4595-4597.
[6] Pons, Anne-Marie, et al. "Microcin E492 is an unmodified peptide related in structure to colicin V." Antimicrobial agents and chemotherapy 46.1 (2002): 229-230.
[7] Lally, Edward T., et al. "The interaction between RTX toxins and target cells." Trends in microbiology 7.9 (1999): 356-361.
[8] Dypbukt, Jeanette M., et al. "Different prooxidant levels stimulate growth, trigger apoptosis, or produce necrosis of insulin-secreting RINm5F cells. The role of intracellular polyamines." Journal of Biological Chemistry 269.48 (1994): 30553-30560.
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