Bacteriophage phi9224 represents a newly discovered member of the Siphoviridae family, infecting Gram-positive Macrococcus bacteria. Mass spectrometry analysis identified nine virion proteins, revealing a genomic modular structure typical of Staphylococcaceae siphophages. However, distinct features such as 31-bp terminal inverted repeats and a short 5’-overhang with variable sequence suggest a unique mechanism of replication and packaging.
The structure of this phage has been determined using single particle cryo-electron microscopy to a resolution of about 3Å. The bacteriophage virion consists of a 65 nm icosahedral capsid (2.96Å) containing the phage genome, a connector between the capsid and the tail, and an approximately 300 nm long tail, which is terminated by a 52 nm long tail tip. The tail structure is composed of the major tail protein forming a hexametric ring arranged into six-entry helix with a twist of 11.5° and 41.67nm rise. The major tail protein was reconstructed to 2.90Å resolution using helical reconstruction algorithm as implemented in CryoSparc [1]. The tail tip of this phase is very inhomogeneous and not visible in cryo-EM data. We show that phi9224 infects Gram-positive Macrococcus bacteria in vitro and we have performed cryo-focused ion beam micromachining (cryo-FIBM) of the phages attached to the bacteria to obtain structural insight into the infection process and the tail tip structure.