Serratia proteamaculans Strain AGR96X Encodes an Antifeeding Prophage (Tailocin) with Activity against Grass Grub (Costelytra giveni) and Manuka Beetle (Pyronota Species) Larvae

A highly virulent Serratia proteamaculans strain, AGR96X, exhibiting specific pathogenicity against larvae of the New Zealand grass grub (Costelytra giveni; Coleoptera: Scarabaeidae), and the New Zealand manuka beetle (Pyronota festiva and P. setosa; Coleoptera: Scarabaeidae), was isolated from a diseased grass grub larva. A 12-day median lethal dose of 4.89 ± 0.92 × 103 cells per grass grub larva was defined for AGR96X, and death occurred within 5–12 days following ingestion of a high bacterial dose. During the infection period, the bacterium rapidly multiplied within the insect host and invaded the hemocoelic cavity leading to a mean bacterial load of 8.2 × 109 cells per larva at 6 days post-ingestion. Genome sequencing of strain AGR96X revealed the presence of a variant of the Serratia entomophila anti-feeding prophage (Afp), a tailocin designated AfpX. Unlike the Afp, AfpX contains two Afp16 tail-length termination protein orthologues and two putative toxin components. A 37-kb DNA fragment encoding the AfpX-associated region was cloned, transformed into Escherichia coli, and fed to C. giveni and Pyronota larvae, causing mortality. In addition, deletion of the afpX15 putative chaperone component abolished the virulence of AGR96X. Unlike the S. entomophila Afp, the AfpX tailocin could be induced by mitomycin C. Transmission electron microscopy analysis revealed the presence of Afp-like particles of varying lengths, and when the purified AfpX tailocin was fed to grass grub or manuka beetle larvae, they underwent similar phenotypic changes to larvae fed AGR96X