Investigating the architecture of a bacteriophage using cryo-EM, SAXS, and X-ray crystallography

Hardy JM1, Dunstan R1, Grinter R1, Pickard D2, Venugopal H3, Belousoff M1, Gordon D2, Lithgow T1 and Coulibaly F1

  1. Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia.
  2. Wellcome Trust Sanger Institute, Hinxton, Cambridgeshire, United Kingdom.
  3. Ramaciotti Centre for Cryo-Electron Microscopy, Monash University, Clayton, VIC, Australia.

YSD1 is a lytic bacteriophage that infects and kills Salmonella enterica serovar Typhi, the causative agent of typhoid fever. YSD1 is a Siphoviridae homologous with the bacteriophage chi, which uses a flagella-dependent infection mechanism. We have shown by electron microscopy (EM) that YSD1 attaches to flagella through its tail proteins. Currently, there are no high-resolution structures of flagella-dependent bacteriophages. To elucidate the structure of YSD1, purified virions were isolated from cultures of Salmonella typhimurium and imaged by cryo-EM. A 2.8 Å x-ray crystallography structure combined with the 4.7 Å EM map revealed a T=7 icosahedral capsid similar to the HK-97 and T7 phages. However, in contrast to HK-97, which uses crosslinking to reinforce the capsid, YSD1 has an additional cementing protein stabilising the icosahedral shell formed by the major capsid protein. Helical reconstruction of the tail produced a 3.8 Å map which revealed a C6 helical tube related to Type VI secretion systems and the tails of T4 and T5 phages. The YSD1 tail is composed of a central beta-barrel domain decorated by two peripheral domains. A beta-sandwich domain, unique to Chi-like phages, has structural similarity with bacterial adherence factors. The C-terminal domain is flexible and not well resolved in the helical reconstruction. The Ig-like fold and organisation of this domain was determined using modelling and small-angle x-ray scattering (SAXS). The determination of the structure of YSD1 gives us an insight into the assembly and evolution of flagella-dependent bacteriophages.