About bacteriophage tail terminator and tail completion proteins: structure of the proximal extremity of siphophage T5 tail

Abstract

Bacteriophages are viruses infecting bacteria. The vast majority of them bear a tail, allowing host recognition, cell wall perforation, and DNA injection into the host cytoplasm. Using electron cryo-microscopy (cryo-EM) and single particle analysis, we determined the organization of the tail proximal extremity of siphophage T5 that possesses a long flexible tail and solved the structure of its tail terminator protein p142 (TrP₁₄₂). It allowed us to confirm the common evolutionary origin between T5 TrP_p142 and other known or putative TrPs from siphophages, myophages, and bacterial tail-like machines, despite very poor sequence conservation. By also determining the structure of the T5 tail proximal extremity after interaction with T5 bacterial receptor FhuA, we showed that no conformational changes occur in TrP_p142 and confirmed that the infection signal transduction is not carried by the tube itself. We also investigated the location of T5 Neck1 or tail completion protein p143 (TCP_p143) and showed, thanks to a combination of cryo-EM and structure prediction using Alphafold2, that it is not located at the capsid-to-tail interface as suggested by its position in the genome, but instead, very unexpectedly, on the side of T5 tail tip, and that it appears to be monomeric. Based on structure comparison with other putative TCPs predicted structures, this feature could not be shared by other TCPs and questions the affiliation of p143 to this family of protein.IMPORTANCEBacteriophages, viruses infecting bacteria, are the most abundant living entities on Earth. They are present in all ecosystems where bacteria develop and are instrumental in the regulation, diversity, evolution, and pathogeny of microbial populations. Moreover, with the increasing number of pathogenic strains resistant to antibiotics, virulent phages are considered a serious alternative or complement to classical treatments. 96% of all phages present a tail that allows host recognition and safe channeling of the DNA to the host cytoplasm. We present the atomic model of the proximal extremity of the siphophage T5 tail, confirming structural similarities with other phages. This structure, combined with results previously published and further explored, also allowed a review and a discussion on the role and localization of a mysterious tail protein, the tail completion protein, which is known to be present in the phage tails, but that was never identified in a phage structure.

Keywords: bacteriophage T5; cryo-electron microscopy; tail completion protein; tail terminator protein.

Fig 1

(A) Scheme of T5 tail organization. The proximal end of the tail (formed of Trp_p142 and TTP_pb6 rings) is framed in red and the upper part of the distal complex (comprising TCP_p143, DTP_pb9, and BHP_pb3) is indicated by a black accolade. (B) Top (upper panel) and side (lower panel) views of the Trp_p142 hexamer as found at the distal extremity of the tail. Two consecutive monomers are not related by a C6 symmetry and are displayed in two different shades of blue. The outer and inner dimensions of the ring are indicated. (C) Superimposition of two Trp_p142 consecutive, non-symmetry related, monomers. (D) Superimposition of two Trp_p142 monomers from native tails (blue) and tails after interaction with receptor FhuA (white). (E) Side view of the proximal extremity of the tail, featuring the Trp_p142 hexamer (in shades of blue) sitting on top of the TTP_pb6 trimer (in cyan). (F) Annotated model of Trp_p142, indicating its main secondary structures. C and N-termini are also indicated. (G) Superimposition of Trp_p142 (blue) and a tail tube domain from TTP_pb6 (yellow). (H) Map of T5 tail structural proteins genes.

Fig 2

Comparison of TrP_p142 with TrPs homologous proteins using DALI pairwise. TrP_p142 is displayed in all panels, colored in dark blue, and superimposed to a selection of homologous proteins of different categories. (A) Siphophages and siphophage-like machines, phage λ (dark green), GTA (light green). (B) Myophages, phage T4 (yellow), phage XM1 (white). (C) Phage-like machines related to myophages: PyocinR2 (red), PVC (orange). (D) Miscellaneous: Type4 fimbrial biogenesis protein PilO from P. aeruginosa (gray).

Fig 3

Isosurface view of the non-sharpened cryo-EM maps of T5 tail proximal extremity, in its native state (A, gray) and after interaction with the bacterial receptor FhuA (B, cyan). Left: side interior view and right: top view. The position and thickness of the top view slice are indicated by black accolades in the side view slice. TMP_pb2 is indicated with red arrows in A and is absent in B. The framed panel in A shows the upper part of the tail tube extremity at a higher threshold, with TMP_pb2 running up to the very end of the tube. (C) Slices of a C3 map of the proximal extremity of the T5 tail tube at different positions, showing TMP_pb2 inside the lumen.

Fig 4

(A) Isosurface view of an unsymmetrized and unsharpened cryo-EM map of the tail tip (side view). The BHP_pb3 trimer is in yellow and the DTP_pb9 hexamer is in orange. The additional density located on the side of the BHP_pb3 trimer and corresponding to the monomeric protein TCP_p143 is framed in black. (B–D) Fit of an AF2 predicted model in the additional density (B: unmodified AF2 model, C: flexible-fitted AF2 model, D: flexible-fitted AF2 model in the segmented map). Different views (from left to right: front view, side view, top slice, bottom slice) are shown. The model in panel B is colored according to its per-residue pLLDT/confidence measure. Color key interpretation is the following: 100 to 90 = high accuracy expected, 90 to 70 = backbone expected to be correctly modeled, 70 to 50 = low confidence/caution, 50 to 0 = should not be interpreted, a strong predictor of disordered regions.

Fig 5

Alphafold2 predictions for different phage TCPs, colored according to their per-residue pLLDT (confidence measure), except for the flexible fitted TCP_p143 (purple). The pLLDT color key interpretation is as in Fig. 2. The bottom panel models are rotated horizontally by 90° compared to the ones of the top panel. Uniprot entries: T5 (Q6QGE0), λ (P03731), SPP1 (O48447), HK97 (E9RJ97), TP901-1 (Q77K21), P2 (P36934), and Mu (Q01261).