The spike tip protein of bacteriophage T4

Reviewer #1 (Public review):

Summary:

Here, Mattenburger et al use structural biology, biochemistry, and genetics to analyze the membrane-attacking end (spike/spike tip) of the contractile injection systems of two DNA phages (P2 and T4). Understanding how a phage tail mediates host recognition and injects DNA into the host is an important question. This manuscript is divided into two stories. First is a biochemical fractionation showing that the fused spike-spike tip protein of P2 (GpV) is translocated into the host periplasm. Second is a somewhat separate story about the spike tip protein of T4 (gp5.4), which is structurally characterized and shown to aid in infection of E. coli with truncated lipopolysaccharides (LPS). I find the suggestion that gp5.4 aids in penetration of the bacterial envelope the most compelling portion of the manuscript, but I find this conclusion to be insufficiently supported, and the presentation could be described as awkward. Further, while the experiments are generally elegant, I believe additional experiments and a discussion to fully connect the two stories of the manuscript would increase impact.

Strengths:

The manuscript is methodologically careful and adds nuance to our understanding of P2 and T4 spike function. The T4 gp5.4 structure is extensively characterized, with crystallography and cryo-EM support. Many experiments are elegant and clever, specifically the P2 periplasmic fractionation and the ex vivo gp5.4 phage reconstitution. If completely supported and explained, the finding that gp5.4 aids in penetration of the bacterial envelope rather than adsorption is compelling.

Weaknesses:

The novelty of the work is somewhat incremental, as phage injection is known to occur into the periplasm and gp5.4 is known to be part of the spike tip (Taylor et al, 2016). The finding that gp5.4 promotes penetration and DNA delivery in strains with truncated LPS is incompletely supported. The gp5.4am phage plaquing data are incompletely explained, and may generate a more modest effect for gp5.4 than is claimed. The P2 results, although well-performed, do not directly support the T4 experiments given the evolutionary divergence between these two phages. Lastly, the overall organization of the manuscript and writing is lacking as (1) the P2 results are presented within the T4 data, (2) many figures are presented out of order, and (3) there is no discussion to contextualize the results for the reader.

Reviewer #2 (Public review):

Summary:

The manuscript provides a very high-resolution crystal structure of the bacteriophage T4 spike gp5-gp5.4 complex and clear evidence of the importance of gp5.4 for the fitness of the phage and its necessity for successful infection of strains of Escherichia coli with truncated lipopolysaccharide. Evidence, or at least speculation, as to what bacterial compounds gp5.4 interacts with would have been welcome.

Strong points:

(1) Very high resolution detailed crystal structure of the gp5-gp5.4 complex.

(2) First proof of the importance of gp5.4 for bacteriophage T4 and by extension, of homologous proteins in other phages.

Weaker points:

(1) Localisation experiments were performed not with protein 5.4 but the homologous gpV from bacteriophage P2.

(2) The exact mechanism was not yet resolved, i.e. to which bacterial component gp5.4 binds.

Reviewer #3 (Public review):

Summary:

The paper describes the structure of gp5.4, the spike tip of phage T4. This structure was released in the PBD in 2013. The paper further investigates the role of this protein in virion assembly, stability, and infection by comparing the behaviour of the WT phage and a phage without the protein, resulting from an amber mutation in the phage genome. A competition assay between the WT and mutant phage shows a clear increase in the fitness of the WT. A further screening of a transposon bank allowed for the identification of a host strain that is resistant to the mutant phage while still sensitive to the WT phage.

Strengths:

(1) Beautiful structure, at very high resolution (1.15 Å).

(2) Very sophisticated microbiology experiments to allow mutant phage characterisation and dissect the role of the spike tip in phage fitness.

Weaknesses:

(1) The paper is very descriptive, and the lack of a general conclusion, not to say discussion, is frustrating. What do the findings of the paper bring to the knowledge of infection? What would be the fate of the spike and tip? A discussion in the context of the data available in the literature would greatly increase the interest of the paper.

(2) Why didn't the authors include the description of the structure of the homologous Pvc10 and PhiKV gp5.4 in complex with gp5ß, which they also solved a while ago?

(3) Because microbiology is sophisticated, special care should be taken to introduce the strains used (both E. coli and T4). E.g. it is still not clear to me what the difference is between the supF and the supD coli strains in terms of mutant phage produced (both should produce T4(5.4am)-gp5.4?).

(4) For the same reason, strains should always be called by the same name.

(5) In some sections, the conclusion seems lost in the description of controls (e.g. in the "The spike is translocated into the periplasmic space during infection" paragraph).

Appraisal:

The authors show that the sharp tip of the membrane-perforating tube of T4 contractile tail contributes to perforating the outer membrane. In particular, this protein is necessary in a host bearing mutated LPS.