Tubulins are essential for the reproduction of many eukaryotic viruses, but historically bacteriophage were assumed not to require a cytoskeleton. Here we identify a tubulin-like protein, PhuZ, from bacteriophage 201φ2-1 and show that it forms filaments in vivo and in vitro. The PhuZ structure has a conserved tubulin fold, with a novel, extended C-terminus that we demonstrate to be critical for polymerization in vitro and in vivo. Longitudinal packing in the crystal lattice mimics packing observed by EM of in vitro formed filaments, indicating how interactions between the C-terminus and the following monomer drive polymerization. Finally, we show that PhuZ assembles a spindle-like array required for positioning phage DNA within the bacterial cell. Correct positioning to the cell center and optimal phage reproduction only occur when the PhuZ filament is dynamic. This is the first example of a prokaryotic tubulin array that functions analogously to the microtubule-based spindles of eukaryotes.

Dynamic instability, polarity, and spatiotemporal organization are hallmarks of the microtubule cytoskeleton that allow formation of complex structures such as the eukaryotic spindle. No similar structure has been identified in prokaryotes. The bacteriophage-encoded tubulin PhuZ is required to position DNA at mid-cell, without which infectivity is compromised. Here, we show that PhuZ filaments, like microtubules, stochastically switch from growing in a distinctly polar manner to catastrophic depolymerization (dynamic instability) both in vitro and in vivo. One end of each PhuZ filament is stably anchored near the cell pole to form a spindle-like array that orients the growing ends toward the phage nucleoid so as to position it near mid-cell. Our results demonstrate how a bacteriophage can harness the properties of a tubulin-like cytoskeleton for efficient propagation. This represents the first identification of a prokaryotic tubulin with the dynamic instability of microtubules and the ability to form a simplified bipolar spindle.DOI: http://dx.doi.org/10.7554/eLife.03197.001

SUMMARY Tubulins are a universally conserved protein superfamily that carry out diverse biological roles by assembling filaments with very different architectures. The underlying basis of this structural diversity is poorly understood. Here, we determine a 7.1 Å cryo-EM reconstruction of the bacteriophage-encoded PhuZ filament and provide molecular-level insight into its cooperative assembly mechanism. The PhuZ family of tubulins is required to actively center the phage within infected host cells, facilitating efficient phage replication. Our reconstruction and derived model reveal the first example of a three-stranded tubulin filament. We show that the elongated C-terminal tail simultaneously stabilizes both longitudinal and lateral interactions, which in turn define filament architecture. Identified interaction surfaces are conserved within the PhuZ family, and their mutagenesis compromises polymerization in vitro and in vivo. Combining kinetic modeling of PhuZ filament assembly and structural data we suggest a common filament structure and assembly mechanism for the PhuZ family of tubulins.