Part:BBa_K3245010

Part Name MccB17

Description:

This part is a full functional gene cluster that produces microcin B17 ( MccB17 ), a kind of antimicrobial peptide. MccB17 belongs to class I microcin, one of the toxic peptides secreted by enterobacteria ( E.coli in this case ). It only works when protease TId D/E ( PmbA ) complex exists which is able to remove the leading peptide to produce mature microcin. The compound displayed antibacterial activity on some strains of E. coli, Enterobacter, Pseudomonas and Shigella.

Usage and biology:

MccB17 inhibits DNA replication by binding with DNA gyrase and induces DNA double-strand cleavage and degradation. The specificity of target bacteria depends on the properties of different gyrases and homologous resistance genes. Microcin genesis requires structural gene ( McbA ), synthase ( McbBCD ) and protease ( TldD/E, outside the original plasmid ). McbE and McbF control efflux, and McbG is responsible for immunity. We introduced this gene cluster to enhance Nissle’s competitiveness and reduce risk of illness caused by some opportunistic pathogens.

Design:

We keep most of the original sequence but adjust several codons to meet RFC10 standard.

Figure 1. Details of The MccB17 gene cluster. It shows the McbA gene product and how it is processed by the microcin synthase (McbBCD) and protease (TldD/E). McbE and McbF control efflux, and McbG is responsible for immunity.

Characterization:

Due to the toxicity of MccB17, its concentration in cells is very low and it’s hard to gain precise data without purification. We used soft agar double-layer technique to measure the inhibition zone and compared the result with common antibiotics such as ampicillin and kanamycin. However, Nissle with MccB17 shows faint effect of inhibiting E. coli DH10B. Further measurement is needed.

Reference

Collin F, Maxwell A. The Microbial Toxin Microcin B17: Prospects for the Development of New Antibacterial Agents. J Mol Biol. 2019;431(18):3400–3426. doi:10.1016/j.jmb.2019.05.050

S. Duquesne, D. Destoumieux-Garzón, J. Peduzzi, S. Rebuffat. Microcins, gene-encoded antibacterial peptides from enterobacteria

Nat. Prod. Rep., 24 (2007), p. 708, 10.1039/b516237h