Bacteria occupy environments where they must compete with many species for limitedresources. One mechanism some bacterial strains employ is contact-dependent inhibition (CDI),which involves toxin delivery upon direct cell-to-cell contact. CDI has been extensively studied,with various toxin types, including DNases, RNases, and ionophores, being identified. Beyondthe inhibitory effect, CDI has been shown to influence gene expression as it was observed togenerate heterogeneity in isogenic bacterial populations. However, little is known regarding thiseffect of CDI, emphasizing the need for further research to deepen our understanding ofbacterial competition and communication.

The aim of this project was to design and assemble genetic constructs that could be used as atool to study and characterise newly discovered CDI toxins. Specifically, the project focused onexamining gene expression changes induced by toxin exposure in isogenic populations.To fulfil the project aim, we constructed chromosomal and plasmid-based reporter constructs,where yellow fluorescent protein (sYFP2) was either transcriptionally fused to target genes ordriven by the promoters of the same genes. These reporter constructs were tested incompetitions against a wide array of CDI toxins and in strains with intracellular overexpressionof toxins. Fluorescence was assessed using flow cytometry and microscopy.

In this study we successfully constructed and tested a chromosomal fruA-sYFP2 reporter,providing a tool for investigating response to toxins. While the construction of additionalreporters proved challenging, the project identified key factors critical for future reporterdevelopment. These include evaluating transcriptional upregulation of target genes prior toconstruct design, understanding how transcript length affects expression levels, and optimizingthe spacer sequence between the reporter genes stop codon and the fluorescent protein’sribosome binding site.