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.

Characterized by Fudan in 2020

We divided this part into two parts: McbABCD and McbEFG.

Method

In order to prove that McbABCDEFG does have an effective antibacterial effect as well as McbEFG can protect the engineered bacteria themselves and help the secretion of antimicrobial peptides more effectively, we designed the following experiment:

We mixed WT E.coli (expressing GFP driven by plac) and E.coli with mcbABCD-mcbEFG-ptetR in different ratios (5:7 20:7 50:7), and measured the OD value of the bacteria two hours after adding an inducer, which can be used to reflect the antibacterial effect of antimicrobial peptides. We followed the same method as above to mix WT E. coli and E.coli with merely mcbABCD, induce and measure the OD value.

Results

Figure1. MEFL/particle for different Px and PxAD when the ratio of mixure(WT:Px=5:7)

Figure2. MEFL/particle for different Px and PxAD when the ratio of mixure(WT:Px=20:7)

Figure3. MEFL/particle for different Px and PxAD when the ratio of mixure(WT:Px=50:7)

We could see an overall decrease of GFP expression in nearly all groups cocultured with Px or PxAD than the control group which only contains WT, indicating that the antimicrobial peptide(MccB17) encoded by mcbABCD does have a negative effect on over microbial. In this case, the living status of WT cells and its function in creating GFP is strongly restricted by the toxic environment, proving that our part mcbABCD has worked successfully.

While inside each paired group, there clearly are a better inhibition effect in Px group than PxAD group, as the MEFL/particle is much lower in the Px group than the PxAD group when driven by certain promoters. This shows that the immunity function of mcbEFG is working successfully, as the E.coli expressing mcbEFG can help the engineered strain to survive with efflux exporting the toxic peptide, as well as better killing off other strains to gain survival advantage.

It is worth noting that when the mixing ratio of WT E. coli and McbABCDEFG-expressing bacteria is 50:7, the OD values of each group are very close to those of the control group (only induced WT E. coli). This shows that when the concentration of the engineered bacteria is very low, the antimicrobial peptides are difficult to exert antibacterial effect and the engineered bacteria have no competitive advantage.

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