Poster Presentation 50th Lorne Proteins Conference 2025

Tailoring tailocins: development of a recombinant expression system for modular phage tail–like bacteriocins (#134)

Jose Benjamin P. Dar Juan 1 , Iain D. Hay 1 , Matthew D. Templeton 1 2
  1. University of Auckland, Auckland, New Zealand
  2. Plant & Food Research, Auckland

The bacteria Pseudomonas syringae pv. actinidiae (PSA) is a phytopathogen that infects Actinidia deliciosa and Actinidia chinensis, commonly known as the green and gold kiwifruit respectively (Vanneste, 2017). In 2010 the ‘biovar 3’ strain of PSA had greatly disrupted the growing New Zealand kiwifruit industry, with export losses by 2014 being estimated at NZ$930 million. While industry has since recovered biovar 3 remains at large with some isolates having acquired resistance to copper, the mainstay of PSA biocontrol, highlighting the need for novel biocontrol agents (Ministry for Primary Industries, 2020; Zhao, Butler, Taiaroa, & Poulter, 2018; Stuart, 2016). A possible solution could come from phage tail-like bacteriocins, or simply tailocins, which are structures derived from prophages co-opted by bacteria as non-replicating bactericidal nanomachines that are believed to be produced to confer a competitive advantage by killing off susceptible strains of bacteria (Scholl, 2017; Ghequire et al., 2015; Nakayama et al., 2000). This is achieved through tail fibers binding to receptors on the cell surface, commonly O-antigen moieties of the lipopolysaccharide that allow the tailocin to form a pore that destabilizes the proton gradient of the cell (Scholl, 2017; Bhattacharjee et. al., 2022). Such a mechanism of action is highly potent wherein a single particle is sufficient to kill a cell. However, recognition of susceptible bacteria is also highly specific which in practice leads to a narrow killing spectrum that limits their application as a biocontrol agent. Our project seeks to address this by developing a recombinant expression system that allows us to express tailocins with alternative tail fibres to alter the killing spectrum of the tailocin. This will be achieved by first confirming the positive transcription factor(s) that regulates the tailocin biosynthetic gene cluster and then cloning the gene into an inducible rhamnose promoter, thereby allowing us to express tailocins on demand. Tentatively, the genes prtN and one encoding a novel R3H domain-like protein appear to induce tailocin expression independent of Mitomycin C, a common inducer of tailocin expression. Further studies intend to replace the native tail fibre and to elucidate the regulatory mechanism of the tailocin biosynthetic gene cluster.

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