Nitrate runoff, or the loss of nitrate nitrogen from agricultural systems, can lead to issues such as unsafe drinking water and overgrowth of aquatic plant life. To combat this issue, we have designed a nitrate-capture system utilizing curli proteins, which are self-assembling amyloid fibers secreted by E. coli that will form a mesh-like structure upon secretion. Our goal is to fuse the nitrate-capturing protein native to cyanobacteria, nrtA, to the curli monomeric unit, csgA. This fusion will be done with the SpyTag/SpyCatcher system, a two-part system that, upon contact, spontaneously forms an isopeptide bond. We are planning our capture system to be a cell-free mesh that can be encapsulated and placed near waterways in agricultural communities. However, we eventually want to modify our construct into a cellular system under the control of an inducible promoter that produces nitrate-capturing curli only in the presence of nitrate.
Our team is located at the University of Illinois at Urbana-Champaign, a school in the midwestern United States surrounded by acres of farmland - primarily cereal crops. Subsequently, agriculture is a major aspect of the surrounding community, which is one of the reasons we chose to combat one of the major global challenges in agriculture - nitrate runoff.
As of 2019, modern agriculture could only sustain about 7 billion of the projected 8 billion+ people in the world, and as a result, global cereal crop production has doubled over the past 40 years [1]. The main method for combatting this issue is increasing the use of nitrate fertilizer. Nitrate is the most limiting factor for plant growth and is an essential nutrient for them. Though these fertilizers have had the desired effect of increasing global crop yields, they have exacerbated the issue of nitrate runoff.
Figure 1. Recent developments leading up to the current issue of nitrate runoff.
Because of these challenges, we decided to instead focus on reducing the negative impacts of nitrate fertilizers by creating a novel nitrate-capture system. Our approach utilizes amyloid-fibers that are naturally produced by E. coli - a cost-effective and environmentally friendly method for a nitrate-capture system.
Our idea was inspired by studies from Tay et al., who created engineered catalytic biofilms to capture heavy metals [2], [3]. Our approach will utilize the Biofilm Integrated Nanofiber Display (BIND) strategy developed by Botyanszki et al. in which the SpyTag/SpyCatcher attachment system is used to site-specifically immobilize a protein onto bacterial nanofibers [4]. The activity of the immobilized protein is independent of the cell - it can remain active even after cell death or if the fibers are unattached from the cell [2], [4]. We aim to first test the ability of our construct to capture nitrate by creating a cell-free engineered nanofiber mesh. Then, we hope to modify our system into a cellular system that can be induced in the presence of nitrate.
An overview of our overall project goals:
1. Fuse a nitrate-binding protein (nrtA) to the curli biofilm produced by E. coli to create a cell-free novel nitrate-capturing system
2. Modify our nitrate-capture system into a cellular system under the control of a nitrate-sensitive promoter
3. Develop a recycling method to repurpose the captured nitrate
Bacterial biofilms are communities of bacteria encapsulated in a secreted matrix of various proteins and polysaccharides. They are usually viewed negatively due to their role in infection and providing bacteria with antibiotic resistance. However, we want to utilize these secreted materials and repurpose them to capture pollutants.
E. coli and Salmonella biofilms contain proteins called curli, which are amyloid fibers that will self-assemble when secreted. The main functional curli monomer is csgA, and when it is expressed and secreted by bacteria it will assemble into a free floating, mesh-like structure that can be further modified [2].
We initially want to test the ability of this self-assembled mesh to act as a scaffold that we can immobilize a nitrate-capturing protein onto. We eventually want to modify this system so that our nitrate-capturing curli will remain tethered to bacteria so we can control curli production with a nitrate-sensitive promoter.
Figure 2. Cartoon of a bacterium displaying secretion of self-assembling curli fibers with the ability to capture various environmental pollutants. Taken from [3].
NrtA is a nitrate-binding protein native to cyanobacteria - autotrophs that are able to capture nitrate and carbon and convert them into cellular biomass. NrtA is the part of the nitrate transport system that initially binds nitrate and is the most abundant protein in the cyanobacteria membrane [5].
We are planning to utilize this nitrate-binding property in our construct.
Figure 3. nrtABCD transport chain native to cyanobacteria. Taken from [5]
The Biofilm Integrated Nanofiber Display (BIND) strategy developed by Botyanszki et al. utilizes SpyTag/SpyCatcher attachment system to site-specifically immobilize a protein onto bacterial nanofibers [4]. It has been used to successfully link a functional amylase protein onto curli nanofibers [4].
This attachment system consists of two parts: the SpyTag, which is a short amino acid sequence, and the SpyCatcher, which is a larger protein. These two components have a high affinity for each other since they come from the collagen adhesin domain (CnaB2) in the fibronectin binding protein, FbaB [6]. Upon contact, the SpyTag and SpyCatcher will form an irreversible bond under a multitude of conditions [6].
Figure 4. Cartoon diagram of the SpyTag/SpyCatcher mechanism. The larger SpyCatcher protein binds with the short SpyTag amino acid sequence upon contact to spontaneously form an isopeptide bond. Adapted from [7].
To create our nitrate-capturing system, we will co-transform 2 plasmids into various curli-producing E. coli: MC4100 and MC4100Z1. The first plasmid will contain the SpyTag, a short amino acid sequence, fused to the main curli protein subunit, csgA protein. The second plasmid will contain the SpyCatcher, a small protein, fused to the nitrate-binding nrtA. When the SpyTag and SpyCatcher come into contact with each other, an irreversible covalent bond is formed, securely attaching nrtA onto the curli biofilm.
Figure 5. Diagram of our proposed csgA-nrtA fusion. The fusion will be done through co-transforming two plasmids: one containing the SpyTag fused to the csgA and the second containing SpyCatcher fused to nrtA.
References
[1] Z.-H. Wang and S.-X. Li, “Chapter Three - Nitrate N loss by leaching and surface runoff in agricultural land: A global issue (a review),” in Advances in Agronomy, vol. 156, D. L. Sparks, Ed. Academic Press, 2019, pp. 159–217. doi: 10.1016/bs.agron.2019.01.007.
[2] P. K. R. Tay, A. Manjula-Basavanna, and N. S. Joshi, “Repurposing bacterial extracellular matrix for selective and differential abstraction of rare earth elements,” Green Chem., vol. 20, no. 15, pp. 3512–3520, Jul. 2018, doi: 10.1039/C8GC01355A.
[3] P. K. R. Tay, P. Q. Nguyen, and N. S. Joshi, “A Synthetic Circuit for Mercury Bioremediation Using Self-Assembling Functional Amyloids,” ACS Synth. Biol., vol. 6, no. 10, pp. 1841–1850, Oct. 2017, doi: 10.1021/acssynbio.7b00137.
[4] Z. Botyanszki, P. K. R. Tay, P. Q. Nguyen, M. G. Nussbaumer, and N. S. Joshi, “Engineered catalytic biofilms: Site-specific enzyme immobilization onto E. coli curli nanofibers,” Biotechnology and Bioengineering, vol. 112, no. 10, pp. 2016–2024, 2015, doi: 10.1002/bit.25638.
[5] N. M. Koropatkin, H. B. Pakrasi, and T. J. Smith, “Atomic structure of a nitrate-binding protein crucial for photosynthetic productivity,” Proc. Natl. Acad. Sci. U.S.A., vol. 103, no. 26, pp. 9820–9825, Jun. 2006, doi: 10.1073/pnas.0602517103.
[6] B. Zakeri et al., “Peptide tag forming a rapid covalent bond to a protein, through engineering a bacterial adhesin,” Proceedings of the National Academy of Sciences, vol. 109, no. 12, pp. E690–E697, Mar. 2012, doi: 10.1073/pnas.1115485109.
[7] S. C. Reddington and M. Howarth, “Secrets of a covalent interaction for biomaterials and biotechnology: SpyTag and SpyCatcher,” Current Opinion in Chemical Biology, vol. 29, pp. 94–99, Dec. 2015, doi: 10.1016/j.cbpa.2015.10.002.
Thanks for the collaborator teams and the sponsor of our university
