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Characterisation of the Enterobacter bugandensis type VI secretion system in microbial antagonism Amy Julia Gail Anderson (24304544) PUREID: 685003384 Enterobacter type VI secretion system The type VI secretion system (T6SS) is a transmembrane nanomachine used by Gram-negative bacteria to inject effectors into neighbouring cells. Effectors can perform various activities and antagonise a range of cell types, including both prokaryotic and eukaryotic cells. Despite being well studied in some bacteria, understanding of the T6SS in Enterobacter species remains limited and underexplored. This thesis focused on a pathogenic clinical isolate of E. bugandensis for in-depth T6SS characterisation in vitro and in silico, with comparative analyses across a curated dataset of E. bugandensis and E. cloacae strains. First, we described that most Enterobacter strains encode T6SS clusters, with all T6SS-positive strains encoding a conserved primary cluster (T6SSi3) and a variable number (0-3) of secondary clusters (T6SSi2-i-iii). These included two new T6SS clusters that have not been previously described. We then investigated the role of the T6SS in microbial antagonism and found that our model isolate produces an offensive T6SS, capable of antagonising both bacteria and yeast. To identify which effectors were responsible for these phenotypes, we then combined bioinformatic and proteomic approaches to identify 26 putative effectors across the genome. We focused on a subset of effectors for functional characterisation in microbial antagonism and identified an evolved VgrG with metallopeptidase domain that displayed a dual role in bacterial and fungal antagonism. A putative- NAD+/NADP+ glycohydrolase, and -pore-forming effector were also described to mediate anti-bacterial activity. Together, this thesis demonstrates that the T6SS is widespread across Enterobacter strains, and can represent a potent, multifaceted weapon for these species. Thesis is embargoed until 31 July 2028. 2026-10-01T16:09:05Z Text Thesis 10.17034/32826305.v1 https://figshare.com/articles/thesis/Characterisation_of_the_i_Enterobacter_bugandensis_i_type_VI_secretion_system_in_microbial_antagonism/32826305 All Rights Reserved Open Access after 2028-07-31