Isolation of an E. coli flagellotrophic Jumbophage SHEFM2K that replicates in cytoplasmic putative assembly areas

The bacteriophage SHEFM2K was isolated from unpasteurised dairy farm milk using a newly isolated E. coli ExPEC strain EcM2K (O23:H8, ST446). SHEFM2K is large contractile-tailed jumbophage with a genome of 348 kb sharing homology with jumbophage from E. coli of the Asteriusvirus genus. SHEF-M2K host range testing indicated that it only makes clear plaques with EcM2K and a sepsis strain from our collection (G34590). Host-ranging assays indicated that it is able to suppress the growth of a range of E. coli strains in liquid culture assays: including EHEC O157:H7, K-12 (MC1000, MG1655) and E.coli B (BL21). TEM images of infection of EcM2K indicated association with flagella-like structures. An E. coli MC1000 mutant lacking the flagellin (fliC) gene was less sensitive to SHEFM2K infection, a phenotype restored by providing fliC in trans. These data illustrate M2K is a flagellotrophic phage that attaches to flagella as part of its infection cycle. We also present cross-sectional TEM images of the SHEFM2K infection cycle showing that it forms putative 'assembly areas' in the host cytoplasm cleared of ribosomes and other material with heads appearing within the periphery before tails appear and lysis occurs. We also present the proteome of mature SHEFM2K phage, highlighting proteins expressed and notable those no detected which might have a role in replication given their predicted function. Overall, we present a preliminary characterisation of a newly isolated jumbophage that interacts with the E. coli flagellum and uncover novel aspects of their biology by identifying an internal assembly area.