Abstract
Plant pathogens cause considerable damage to crop production resulting in global food
shortages and severe economic losses. There is growing restriction for the use of
agrichemicals, such as copper and antibiotics, in response to escalating resistance and
environmental accumulation. Therefore, there is a desperate need for new, alternative,
environmentally friendly antimicrobial agents. Bacteriophages (phages) naturally
predate bacteria and thus can be harnessed as biocontrol agents. An alternative strategy
is the use of phage-derived enzymes, including endolysins. These enzymes degrade the
peptidoglycan of the cell wall and natively act from the “inside-out”. Applying them to
the outside of bacteria as antimicrobials has shown efficacy against gram-positive
bacteria, however the additional outer membrane of gram-negative bacteria impedes the
endolysin from accessing the peptidoglycan. Therefore, current research majorly focuses
on approaches to enhance the ability of endolysins to overcome the gram-negative outer
membrane. These strategies include the use of chemical membrane permeabilisers and
the engineering of fusion proteins incorporating endolysins, antimicrobial peptides and
phage-derived receptor binding proteins. This thesis investigates the use of phage-
derived proteins against the causal agent of bacterial canker on kiwifruit; the gram-
negative bacterium Pseudomonas syringae pv. actinidiae (Psa). We describe and
investigate the mechanism of an antimicrobial synergy between citric acid and the
endolysin from the phage ⏀Psa374 that is effective at reducing copper resistant Psa in
vitro. We then demonstrate the development of novel chimeric phage-derived proteins
incorporating additional phage proteins such as tail fibers, baseplates, tail spikes, holins
and virion-associated lysozymes. By generating and screening large numbers of variant
fusion proteins we produced a lead variant, L4E10, that utilises a modular endolysin and
a lipase, which displays moderate exogenous antibacterial activity that is specific to
Pseudomonas syringae pathovars. Finally, we developed a vector, containing
Escherichia coli SlyD, which can be co-expressed to enhance the soluble expression and
purification of aggregation-prone proteins; including endolysin - tail fiber fusions.
Overall, this thesis evidences the potential of endolysin fusion proteins as alternative
antimicrobials against gram-negative phytopathogens and explores the prospects of
further phage proteins as an untapped antimicrobial reservoir.