Our team is located in California, one of the largest agricultural states in America. Just an hour away from our high school, the iconic Julian apple orchards, among other pome fruit orchards, help fuel our home state’s economy. However, with the fire blight disease continuing to threaten this prosperity within our own community, we decided to set out ourselves to extinguish this “flame”.
While looking for a treatment to combat fire blight, we focused primarily on treatments with antibacterial properties including bacteriophages, glycohydrolase, and lactoferrin; however, these options were eventually rejected due to their lack of feasibility and/or antimicrobial activity against “helpful” bacteria.
In the end, our team decided on darobactin A, mainly due to its exhibition of specific antimicrobial activity against Gram-negative bacteria, which have a double-layer cell membrane that many other antibiotics have difficulty breaching (Groß et al., 2021). Darobactin is capable of specifically combating only gram-negative bacteria such as E. amylovora because it inhibits the protein BamA (Imai et al., 2019); Only gram-negative bacteria possess the five-protein BAM complex, which is responsible for the folding and insertion of nascent β-barrel OMPs in the outer membrane (Han et al., 2016).
As antibiotic resistance is currently a major issue existing within other fire blight treatments and darobactin’s target of a specific protein protects our project’s vulnerability to causing cross-resistance, we decided that darobactin, a relatively novel compound, would be the best possible choice (Wu et al., 2019).
Our proposed solution to combating fire blight is to produce darobactin in a bacterium, which will be applied onto the plant, with its darobactin production induced only when necessary. To achieve this, our team has chosen to model our solution in E.coli. Though E.coli is a gram-negative bacteria (and therefore darobactin exhibits antimicrobial activity toward it), we have acquired the sequence of a mutated version of the bamA gene that confers resistance to darobactin from the Lewis Lab at Northeastern University, as well as the plasmid they used to produce darobactin (Imai et al., 2019). For proof of concept testing, we were also graciously given purified darobactin from the same lab.
Our team made our own changes to the given plasmid, pNB03-darA-E, by adding the mutated bamA gene and our chosen inducible promoter: the lac promoter. Transcription of the lac operon is triggered by the lactose metabolite Isopropyl β- d-1-thiogalactopyranoside (IPTG), giving us the ability to switch darobactin production on and off. In our future implementation in the agricultural setting, the use of an infection induced promoter would be added in place of the lac promoter (see promoter page). The mutated bamA gene, in turn, will be expressed when the promoter is induced; when the promoter is not induced, the gene will not be expressed, leaving only the wildtype BamA that will make the bacterial vector susceptible to the leftover darobactin in the environment.
However, we encountered some limitations in the design of our project. Though we chose to model our darobactin production in E. coli, we recommend a strain of Pantoea agglomerans for a more practical solution. Unlike E. coli, P. agglomerans naturally colonizes fruit trees and is able to survive off of a tree’s nutrients, making it a more feasible choice for real-life application. In our case, because P. agglomerans is much more complicated than E. coli and harder to access, modeling in E. coli was more feasible as a high school team with limited resources. Despite this, we designed an additional plasmid for use in P. agglomerans.
Some ethical concerns included biosafety and antibiotic overuse. We attempted to diminish both these concerns by limiting the amount of darobactin released into the natural environment by using an infection induced promoter, such as the pelE promoter, in the place of the lac operon in the P. agglomerans plasmid. We have also presented a few other promising promoters that need further research regarding specificity to E. amylovora. These promoters will add specificity to the product by initiating the production of darobactin during a plant infection. Furthermore, darobactin has been proven to be non-toxic against common symbiotic gut bacteria and human cell lines, reducing safety concerns of darobactin on produce (Seyfert et al, 2023). In addition, the plasmid that we hope to design using P. agglomerans EH318 has found to be effective against fire blight and the derivatives are safe for fire blight infections (S. A Wright et al.). Additionally, the strain E325 has been used in Bloomtime, an effective treatment against Erwinia herbicola (US EPA report).
Han, L., Zheng, J., Wang, Y. et al. Structure of the BAM complex and its implications for biogenesis of outer-membrane proteins. Nat Struct Mol Biol 23, 192–196 (2016). https://doi.org/10.1038/nsmb.3181
Imai Y, Meyer KJ, Iinishi A, Favre-Godal Q, Green R, Manuse S, Caboni M, Mori M, Niles S, Ghiglieri M, Honrao C, Ma X, Guo JJ, Makriyannis A, Linares-Otoya L, Böhringer N, Wuisan ZG, Kaur H, Wu R, Mateus A, Typas A, Savitski MM, Espinoza JL, O'Rourke A, Nelson KE, Hiller S, Noinaj N, Schäberle TF, D'Onofrio A, Lewis K. A new antibiotic selectively kills Gram-negative pathogens. Nature. 2019 Dec;576(7787):459-464. doi: 10.1038/s41586-019-1791-1. Epub 2019 Nov 20. Erratum in: Nature. 2020 Apr;580(7802):E3. doi: 10.1038/s41586-020-2063-9. PMID: 31747680; PMCID: PMC7188312.
Sebastian Groß, Fabian Panter, Domen Pogorevc, Carsten E. Seyfert, Selina Deckarm, Chantal D. Bader, Jennifer Herrmann, Rolf Müller: Improved broad-spectrum antibiotics against Gram-negative pathogens via darobactin biosynthetic pathway engineering. Chem. Sci. 2021; doi: 10.1039/D1SC02725E
Seyfert, C. E., Müller, A. V., Walsh, D. J., Birkelbach, J., Kany, A. M., Porten, C., Yuan, B., Krug, D., Herrmann, J., Marlovits, T. C., Hirsch, A. K., & Müller, R. (2023). New genetically engineered derivatives of antibacterial Darobactins underpin their potential for antibiotic development. Journal of Medicinal Chemistry, 66(23), 16330–16341. https://doi.org/10.1021/acs.jmedchem.3c01660
Wu R, Stephenson R, Gichaba A, Noinaj N. The big BAM theory: An open and closed case? Biochim Biophys Acta Biomembr. 2020 Jan 1;1862(1):183062. doi: 10.1016/j.bbamem.2019.183062. Epub 2019 Sep 11. PMID: 31520605; PMCID: PMC7188740.