Investigation of the Tail Fibres and Tail Fibre Assembly Proteins of Contractile-tailed Phages

The tail fibres of long-tailed phages are complex elongated protein assemblies capable of specific recognition of bacteria during the first step of viral infection. Correct assembly of these structures is crucial for successful phage propagation and often requires a phage-encoded tail fibre assembly (Tfa) protein. In Escherichia coli phage T4, the formation of the distal portion of tail fibres requires a Tfa (product of gene 38) which is absent from mature particles. Despite the essential role of Tfa proteins for many diverse phages, only a few studies have addressed their functional mechanisms. Here, I investigate the tail fibre and Tfa of E. coli phage Mu. I demonstrate that Tfa forms a stable complex with the tail fibre, and I present a 2.5 Å resolution X-ray crystal structure of this complex. The Tfa:tail fibre complex is shown to bind the phage baseplate wedge and lipopolysaccharide, the outer membrane receptor for this phage. My in vitro and bioinformatics studies reveal that Tfa proteins are comprised of two domains: a non-conserved N-terminal domain that binds to the fibre and a conserved C-terminal domain that likely mediates fibre oligomerization and assembly. I propose that the key role of Tfa proteins is to initiate multimerization and assembly at the C-terminus of fibres, ensuring that their intertwined and repetitive structural elements come together in the correct sequence. Surpringly, I also detected Mu Tfa in the mature particles of Mu. In addition, I discovered that the Tfa proteins of phages Mu and E. coli phage P2 bound to lipopolysaccharide. Despite sharing 93% sequence identity these Tfa proteins displayed different LPS-binding preferences, consistent with those of full phage particles. In summary, I conclude that Mu Tfa is required for tail fibre folding and oligomerization as was seen for phage T4. In contrast to T4, however, Mu Tfa binds stably to the tail fibre and stays bound even in the mature particle. This observation coupled to the demonstrated LPS-binding activity of Mu and P2 Tfa suggests that Tfa proteins may play a role in determining host specificity. Thus, the function of “chaperone” may not apply to many Tfa proteins.Ph.D.2021-03-21 00:00:0