Insights into the assembly and biology of the Serratia entomophila Anti-feeding Prophage

Abstract

The Serratia entomophila antifeeding prophage (Afp) forms a phage-tail-like particle that acts on the New Zealand grass grub, Costelytra zealandica. An optimised expression and purification protocol for the Afp and its derivatives was developed. The ability to purify the Afp in conjunction with bioassays enabled the 3 day LD50 to be defined as approximately 500 Afp particles required to cause a cessation of feeding activity by the C. zealandica larva. Genetic manipulation allowed for construction of Afp variants. Genes (afp1-18) encoding components of Afp were expressed and their products purified allowing morphological assessment of the products by transmission electron microscopy (TEM). Expression of afp1-15 resulted in the formation of a nonsheathed structure termed the tube-baseplate complex (TBC), composed of an irregular-length tube attached to a baseplate with associated tail fibers. Expression of afp1-16 produced mature, normal-length Afp particles, whereas co-expression of afp16 with afp1-15 in trans resulted in the formation of aberrant Afp particles of variable lengths. A C-terminally truncated Afp16 mutant resulted in a phenotype intermediate between mature Afp and TBC. Analysis of TEM images of purified Afp16 revealed a hexameric ring-like structure similar to that formed by the gp3 of phage T4 and gpU of phage λ. The addition of purified Afp16 to Afp unravelled by acidic treatment resulted in the formation of shorter tubes when sample pH was adjusted to 7 than those formed in the absence of Afp16. These results suggested that Afp16 terminates tube elongation and is involved in sheath formation. Assessment of purified Afp particles derived from pAF6 clones bearing transposon insertions in the ORF afp14 resulted in the production of mature Afp of shorter than regular lengths. The resultant lengths corresponded with the predicted length of the translated protein up to the transposon insertion site, suggesting that Afp14 is a ruler protein determining the length of the Afp. Subjecting the Afp and TBC to a variety of chemical treatments resulted in structural alterations. Bioinformatic analysis and comparison of Afp and TBC protein compositions in combination with the observed mode of Afp length control allowed the development of a model for Afp assembly. Keywords: Serratia entomophila, Anti feeding prophage, Afp, tailocin, Afp16, tail length termination, tail termination protein, TrP, Afp14, ruler protein