PREreview of A prophage-encoded anti-phage defense system that prevents phage DNA packaging by targeting the terminase complex

The manuscript by Azulay et al. investigates the cooperative interaction between Listeria monocytogenes (Lm) strain 10403S and its prophage ɸ10403S. The authors identify and characterize a novel anti-phage defense system that is encoded in the genome of the prophage focusing on the ORF65 protein renamed TerI (for terminase inhibitor). The study shows that TerI inhibits phage reproduction by targeting the phage terminase complex of invading phages, thus preventing the packaging of phage DNA. Azulay et al. also discover two other prophage encoded proteins that provide the phage with immunity against its own TerI protein and thus enable packaging of its own genetic material. This preprint is well-written and contains high-quality data that advances the field of phage biology and anti-phage defense systems. We provide below some suggestions to strengthen the methodology, and to increase the clarity in the interpretation of the results.

Major comments

A loading control is required in Figure 1B, 2F, 2G, and 4D to verify that the amount of protein entered into each well is the same either using an antibody against a protein that does not change or using Coomassie or Ponceau stain of the membrane. Additionally, relative quantification (to the loading control) of immunoblots would be helpful to measure the loss of ORF65 co-sedimentation in the ΔterSL mutant (Figure 2G) to strengthen the finding that ORF65/TerI interacts with TerS. For Figure 2F, a quantification of the immunoblot, ideally including a dilution series, would further support the statement that both the bacterial strains, expressing ORF65 or not, produced or released a similar number of capsids.

Page 11: “ectopic expression of each of these genes alone (i.e., without TerI expression) did not affect virion production, as compared to WT bacteria, suggesting that the encoded proteins play a specific role in neutralizing TerI.” In Figure 3D, the ectopic expression of LMRG_01518 shows a large standard deviation reaching about 50%WT in PFU, suggesting that overexpression of LMRG_01518 may impact virion production. We recommend showing individual data points of each replicate and to provide a possible explanation for this high standard deviation.

Page 12: “Altogether, these findings indicated that TerI is fully active during 10403S induction and that LMRG_01518 and LMRG_02984 counteract its activity, functioning as self-immunity proteins that allow 10403S DNA packaging and virion assembly”. However, only LMRG_02984 directly interacts with TerI and not LMRG_01518 (Figure 3H). This data may explain the stronger phage ɸ10403S rescue consistently observed when LMRG_02984 is present compared to LMRG_01518 (Figure 3E). There is a question as to whether the results, as currently reported, fully support the direct interaction of LMRG_01518 with TerI. The study could incorporate a BACTH assay between LMRG_02984 and LMRG_01518 to support that conclusion, or the text could be revised to ensure the conclusions tightly align to the findings currently included (e.g. the model in Figure 5 depicts direct interaction between anti-TerI1(01518) and TerI).

The discussion, while well-written, reads more like a minireview and could focus more on the findings. It could be expanded by elaborating more on the data presented and how it supports the model presented in Figure 5. Of particular interest is the discussion of LMRG_01518 inhibiting TerI function although not interacting with TerI directly. Other interesting points to discuss include the specificity of the TerI/anti-TerI1, I2 system, could the invading phage encode anti-TerI as well? How about the TerI/TerS specificity, from the data presented, it seems that TerI is able to inhibit TerS from different phages. Is there anything known about the function of LMRG_01518 and LMRG_02984 besides their role as anti-TerI? On Figure 3C, it seems that expression of LMRG_01517 largely decreases PFU compared to control, this result could be discussed.

We realize it is beyond the scope of this study, but it would be of interest to perform a BLAST search with terI. This novel anti-phage defense system might be used by other temperate phages in other bacteria. It would be interesting to see how many other phages use this system. Or perhaps this is something exclusive to the Lm phages?

Minor comments

- Page 6: could you explain the choice of not using the his-tagged ORF65 from Figure 1E and on. We understand this may be technically difficult due to lack of specific antibodies against TerI and anti-TerI1 and 2, but something that was not obvious to us was whether the ectopic expression of ORF65 resulted in overexpression compared to endogenous levels in WT and whether exogenous infection leads to anti-terI1 and anti-terI2 expression. Figure 1F, we would have expected higher PFU than WT in the ATG*orf65 if ORF65 is present in WT to prevent virion production, and Figure 1G, we would have expected some level of PFU if anti-TerI are present to counteract TerI.

There is a sentence that could be clarified “To this end, we infected Lm bacteria cured of ɸ10403S and deleted the comKgene with free particles of ɸ10403S in the presence or absence of orf65 ectopic expression (i.e…).” Consider breaking it into two: “We made ΔcomK Lm bacteria cured of ɸ10403S (Lm Δɸ10403S-ΔcomK). We then infected Lm Δɸ10403S-ΔcomK with ɸ10403S particles in the presence or absence of orf65 ectopic expression…”

- Page 11: could you clarify the following sentence “cro (corresponding to the first early gene)” and define the acronym cro in the first instance. It is not clear to us whether cro itself is considered an early gene as it represses early genes.

- Methods: could you describe how the ATG*orf65 mutation was exactly made and explain the choice of this mutation as opposed to deleting the Shine-Dalgarno sequence of the rpsD promoter (if it contains one). Could you also explain why the mutagenesis of the -10 box of the orf65 promoter involved 5 bases and not less.

- Figure 1A (and 3A): LMRG_01515 is labeled as LMRG_1515, include the missing zero in the locus number.

- Figure 1C, 1E, 2C, 2D: plot growth curves on a log scale rather than linear as it is standard in the field.

- Figure 1F, 3C, 3D: y-axis of PFU (% WT) needs to be consistent and should be put on a linear scale rather than a logarithmic scale as it is standard in the field. Provide number of replicates in the panel (such as N = 3).

- Figure 1H: clarify which ones are lytic or temperate (prophages) in the panel by adding labels below the phage names. Also, keep consistent the nomenclature of the phages in the text to match figures (phage names missing phi symbol in text).

- Figure 2F: could you explain the choice of quantifying attP rather than attB.

- Figure 2E: increase scale size and keep panel sizes consistent and aligned.

- Figure 2G: explain the low level of ORF65 detected in the ΔΦ pellet in the first panel. Are the ΔterS and ΔterL mutants available, it would be of interest to test these as well to strengthen the BACTH assay in Figure 2H showing direct interaction with TerS but not L. This may be technically challenging, but all samples could be run on one gel to produce one blot, if possible, for ease of comparison.

- Figure 3: for consistency in the panels presented, the nomenclature used to describe LMRG_02984 and LMRG_01518 should not be changed to anti-TerI1 and anti-TerI2.

- Figure 3A: make the phage genome image larger and include description in the legend.

- Figure 3H: LMRG_01518 was included as part of the negative controls.

Maddy McPherson and Octavio Origel (Indiana University Bloomington) - not prompted by a journal; this review was written within a Peer Review in Life Sciences graduate course led by Alizée Malnoë with input from group discussion including Sally Abulaila, Kim Kissoon, Michael Kwakye, Madison McReynolds, Mandkhai Molomjamts, Habib Ogunyemi and Ren Wilson.

Competing interests

The authors declare that they have no competing interests.