Project Description
Bacterial pathogens cause many of the most devastating plant diseases of the modern age: fire blight of apples and pears, Pierce’s disease in grapes—shown in Figure 1—and crown gall disease, just to name a few (Mansfield et al., 2012). The scourge of plant pathogens in agriculture has affected not only the livelihoods of farmers but also the food supply. Globally, plant pathogens cause roughly $220 billion in crop yield loss every year (FAO, 2017); the potato pathogen Ralstonia solanacearum alone is responsible for over $1 billion in annual agricultural losses (Mansfield et al., 2012).
Figure 1. Leaves on a grapevine showing characteristic symptoms of Pierce’s disease caused by infection with the bacterium Xylella fastidiosa. Source: OSU Extension.
This year, the UT Austin iGEM team is tackling the problem of bacterial plant pathogens in agriculture by engineering protective strains of bacteria (our chassis) to secrete small antimicrobial peptides known as microcins. These peptides are specifically selected to kill the pathogen of interest and have a relatively narrow target spectrum (Parker & Davies, 2022). Our goal is to create a modular system in which microcins targeting different plant pathogens can be easily swapped out with each other for secretion by the chassis, enabling the rapid engineering of multiple biocontrols that each target a specific pathogen.
Current control methods for preventing infection, such as antimicrobial pesticides and insecticides, have many negative impacts and are ultimately unsustainable. Long-term use of antimicrobial pesticides may inadvertently select for resistance in pathogenic bacteria while also killing off beneficial plant symbiotic microbes, and pesticide run-off can cause serious environmental damage as well as threaten human health. Biocontrols, however, are a less-explored but increasingly promising solution to plant pathogen control. While already used in agriculture to a small extent, bacterial biocontrols only make up approximately 1% of current plant pathogen control methods (Lahlali et al., 2022).
Our team’s key goal is to demonstrate that we can engineer a modular system that enables a non-pathogenic bacterial chassis to kill a plant pathogen of interest, thus acting as a biocontrol. In order to inhibit the proliferation of target pathogens, we are engineering our chassis to secrete small antimicrobial peptides known as microcins—a subclass of bacteriocins—that can ideally kill the pathogenic bacterium before it severely infects the plant. These microcins act as molecular ‘weapons’ that the chassis can use against the pathogenic target strain by secreting them outside of the cell. But why microcins?
Currently characterized microcins are highly specific in their bactericidal activity, and this is thought to make it more challenging for target species to develop resistance (Parker & Davies, 2022). This specificity of microcins is generally attributed to their evolution as a weapon of interbacterial competition, as it has been observed that bacteria within the same niche (often close relatives) are the most likely to produce active microcins against one another (Cole et al., 2022). Because of this, microcins generally have a narrow target spectrum and would likely produce less disturbance to the overall plant microbiome than traditional antimicrobial compounds.
To ensure that microcins produced by our chassis can exit the cell to act against their pathogen targets, we have chosen to base our design on a characterized two-plasmid microcin secretion system (Kim et al., 2023). In the original system, one plasmid is a microcin expression plasmid that expresses E. coli microcin V while the other is a secretion system plasmid known as pSK01 (Kim et al., 2023). We specifically redesigned the original microcin expression plasmid to be a modular system. This allows our biocontrol chassis to secrete a microcin of our choosing when it is transformed with pSK01 and our modular microcin expression plasmid.
The first plasmid—labeled as plasmid 1 in Figure 3—contains coding sequences for the type I secretion system proteins CvaA (a membrane fusion protein) and CvaB (C39 peptidase domain-containing ABC transporter) under a constitutive promoter (Kim et al., 2023). The positions of these proteins in the membrane are displayed in Figure 2. The TolC exit channel protein is not expressed from pSK01 because this receptor is already present in the genomes of most Gram-negative bacteria (Kim et al., 2023).
The second plasmid—plasmid 2 in Figure 3—contains the coding sequences for a microcin and its associated immunity protein, the latter of which prevents the microcin from killing the cell that produces it. This plasmid includes a short signal peptide (labeled SP in Figure 2) sequence upstream of the microcin, which binds to the cognate type I secretion system and is subsequently cleaved off during microcin export. There are two variants of this second plasmid—one version places the microcin and immunity protein under a constitutive promoter, while the other instead uses an inducible promoter system for regulation of microcin transcription.
Figure 2. Schematic of the microcin V type I secretion system in E. coli. From Kim et al., 2023.
