The structure of bacteriophage phi9224

Zuzana Hlavenkova1, Ivana Mašlanová3, Tibor Fúzik2, Jiří Nováček1, Roman Pantůček3

1Central European Institute of Technology, Masaryk University, Brno, Czech Republic

2Structural Virology, Central European Institute of Technology, Masaryk University, Brno, Czech Republic

3Department of Experimental Biology, Faculty of Science, Masaryk University, Brno, Czech Republic

 

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.

1.         Punjani, A., Rubinstein, J., Fleet, D. et al. cryoSPARC: algorithms for rapid unsupervised cryo-EM structure determination. Nat Methods 14, 290–296 (2017). https://doi.org/10.1038/nmeth.4169