<p>To lay the foundation for a new avenue of research into engineering nitrogen-fixing endosymbionts, we investigated protein transport to B. bigelowii’s symbiotic partner UCYN-A (nitroplast) and implemented proof-of-principle designer endosymbiosis experiments.
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<divclass="h1">Background</div>
<divclass="h1">DRY LAB</div>
<p>The Netherlands has been facing a pressing <strong>nitrogen crisis</strong> for several years. This crisis is largely attributed to the <strong>agriculture sector</strong>, with over 80% of ammonia (a nitrogenous compound) emissions coming from manure <ahref="#cite1"style="color: #185A4F;">[1]</a> and chemical fertilizers <ahref="#cite2"style="color: #185A4F;">[2]</a>.</p>
<p>The majority of proteins within mitochondria and chloroplasts are nuclear-encoded – they are expressed by the host and are imported into the organelle. Proteins meant for the organelle are usually marked by a targeting sequence at one end, also known as a transit peptide, which directs the protein to its destination after which it is cleaved. </p>
<p>The over-use of fertilizers has a detrimental effect on the environment through the deposition of excess nitrogen oxides and ammonia in the ground, excessively enriching the environment with nutrients promoting uncontrolled plant and algal growth, or eutrophication, a form of nutrient imbalance <ahref="#cite3"style="color: #185A4F;">[3]</a> that negatively impacts the local biodiversity. This highlights the need of the hour: <strong>tackle the nitrogen crisis without negatively affecting food production</strong>, which still depends highly on fertilizers.</p>
<p>This is no different with UCYN-A: Coele et al [1] in their 2024 study used proteomics to find proteins encoded by the host and imported into the nitroplast. Upon examining these protein sequences, they noticed that many of them possess characteristics of organellar import – most of them possess a C-terminal 120 amino acid extension compared to their orthologues. This extension is reminiscent of targeting sequences known to exist in mitochondrial [2] and chloroplastic [3] imported proteins. They termed the putative targeting sequence uTP (UCYN-A Transit Peptide, with lowercase “u” to differentiate it from uridine triphosphate).
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<p>Our investigations began with an in-depth computational analysis of B. bigelowii’s proteome [1, 7] to identify potential signals marking proteins for import into UCYN-A. Based on these results, we designed fluorescent protein-transit peptide constructs for expression in model organisms to show that the identified signals indeed localize to UCYN-A. To pave the way for transplanting the nitroplast into new organisms, we also explored the feasibility of physically inserting UCYN-A into a new host by attempting cell fusion experiments. Furthermore, we successfully established a culture of B. bigelowii and tested a new protocol for isolating UCYN-A. These experiments collectively aim to elucidate the mechanisms of UCYN-A's endosymbiotic relationship and lay the groundwork for future engineering of nitrogen-fixing symbionts into new host organisms.</p>
