Reversible conjugation of a CBASS nucleotide cyclase regulates bacterial immune response to phage infection

Prokaryotic antiviral defence systems are frequently toxic for host cells and stringent regulation is required to ensure survival and fitness. These systems must be readily available in case of infection but tightly controlled to prevent activation of an unnecessary cellular response. Here we investigate how the bacterial cyclic oligonucleotide-based antiphage signalling system (CBASS) uses its intrinsic protein modification system to regulate the nucleotide cyclase. By integrating a type II CBASS system from Bacillus cereus into the model organism Bacillus subtilis, we show that the protein-conjugating Cap2 (CBASS associated protein 2) enzyme links the cyclase exclusively to the conserved phage shock protein A (PspA) in the absence of phage. The cyclase–PspA conjugation is reversed by the deconjugating isopeptidase Cap3 (CBASS associated protein 3). We propose a model in which the cyclase is held in an inactive state by conjugation to PspA in the absence of phage, with conjugation released upon infection, priming the cyclase for activation.

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In the meantime we hope that you find our referees' comments helpful.In this study Krüger et al. describe the reversible conjugation of a CD-NTase (cyclase) to the ubiquitous phage shock protein (PspA) and propose a model by which this conjugation plays a role in the sequestration of the cyclase to limit spurious activation of the Type II CBASS system in the absence of phage infection.They first show the genetic and catalytic requirements of the B. cereus CBASS for protection against phage predation in the heterologous B. subtilis host and further show the unexpected finding that this cyclase is conjugated specifically to the B.subtilis PspA protein in a Cap2 dependent manner.The Cap3 enzyme is also shown to scarlessly proteolyze the cyclase-PspA conjugate in vitro and in vivo, and this activity occurs late during infection with phage Groe23.The cyclase in vitro appears to be in a state of reduced activity, regardless of its conjugated state, in vitro and inactive in vivo in the absence of phage.This suggests that a critical condition to activate the cyclase during infection remains to be discovered, and the authors suggest based on in vitro cyclase assays this could be related to the abundance of intracellular nucleotides (e.g., ATP).Heroic efforts were made to demonstrate the utility of the PspA-cyclase conjugate in vivo, but the physiological role of this interaction remains enigmatic, with no conclusive evidence demonstrating this interaction between the host PspA and the CBASS cyclase is necessary for defense against phage by the Type-II CBASS.The expansion of these findings to include additional phage infection experiments (e.g., Groe26) validating the delayed activation hypothesis, demonstration of cyclase conjugation to diverse PspA substrates (not only B. subtilits PspA), as well as testing the CBASS antiphage activity under control of its native promoter (B.subtilis host is fine) would greatly improve the significance of these findings.
I want to commend the authors for their hard work, and I implore them to please read the following comments with the knowledge that they are meant to be helpful and written with a voice of curiosity and encouragement.There is no malice intended by any language that follows and in no way are they meant to be combative, rude, or demeaning.It is likely that some of these ideas and observations presented may reflect my own misinterpretation of data or text, despite my best efforts to be thorough.Please know that I respect your time, effort, and diligence in preparing this manuscript and thank you for reporting these interesting results.

Primary Comments:
The characteristics of both plaquing and planktonic growth of B. subtilis Δ6 + CBASS challenged with Goe23 are dramatically different from the other phages tested (Figs. 1B & 1C,Ext Data Fig 2).The Goe23 plaquing phenotype and the crash in OD (~3-5h) could be indications of slow activation of CBASS that is specific to this phage, as these are not seen in similar challenges with the three other phage.Because the delayed time to activation (~ 30 min) of this CBASS is an important conclusion of this study, it is important to demonstrate this is not a phenomenon specific to Goe23 infection alone (Figs. 4b,d,e and Ext. Figs. 6 and 7) (i.e., is delayed Cap3 deconjugation/activation of the cyclase only happening with Goe23).Additional temporal experiments (Similar to those performed in Ext.Figs. 6 and 7) should be performed using at least one additional phage.Perhaps Goe26 might be the most reasonable follow-up phage to use as both planktonic and plaquing data are currently presented, which could ease the burden of these experiments.Lines 253-254, "The low sequence conservation between PspA homologs may explain why Cap2 requires flexibility in target recognition (Extended Data Fig. 5c)".The in vitro assay is a neat tool and I commend the authors for the monumental task of purifying all these components and establishing a condition that recapitulates some of the aspects observed in their in vivo conditions.However, the in vitro condition likely has some substantial artifactual activity where the Cap2 enzyme appears more promiscuous in its conjugation of the cyclase to alternative substrates/residues (e.g., high MW complexes of Cap2-cyclase in Fig. 3a not seen in vivo, Figs.2a, 2d, Extended Data 3a, Extended Data Fig. 9a).However, in vivo results demonstrate cyclase-specific peptides are found only on residues K147 and K220 (Fig. 2b, Lines 176-177, Lines 332-333).To suggest that there is flexibility in target recognition by Cap2 in vivo activity to facilitate its ability to work with diverse PspA substrates (i.e., function in diverse bacterial hosts), the authors need to perform similar purifications and Western blots as those presented in Fig. 2d using strains containing the K147R and K220R variants of the B. subtilis PspA and demonstrate whether comparable alternative Lys residues on PspA are truly utilized as substrates in vivo.It is also important to assess the capacity of Cap2 to conjugate the cyclase to B. cereus PspA, which is presumably the "natural" substrate, and determine the locations of preferred Lys residues Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work.The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
for cyclase conjugation.
In Fig. 4d, use of either of the Cap2 inactive Cys->Arg variants (Figs. 1b,c,5a) is important to demonstrate the utility of conjugation and would be more convincing than the double CBASSΔCap2ΔCap3 currently presented (i.e., the variants are a precision tool for addressing the role of conjugation while the double mutant is a hammer).If it is impractical to repeat these experiments with the cap2 variants, it is important to justify the use of the double knockout mutant in lieu of these superior strains (this is also applicable to Fig. 2d, but more critical to address in Fig. 4d where cyclase activity is explored in vivo).
In conjunction with the previous comments, would the authors please double check that in generating the double cap2 cap3 deletion strain (e.g., Fig. 4d) an unintended catalytically inactive chimeric cyclase has not been created (i.e., the native cyclase stop codon is preserved) and the Nuc-SAVED effector is still likely to be functional (i.e., start codon is preserved, a putative ribosome binding site exists, and transcription of the effector is unlikely to be interrupted.. basically check for any obvious indications of polar effects on the effector).There is overlap between the final nucleotide of the cyclase stop codon and the first nucleotide of the cap2 start codon and the Nuc-SAVED effector overlaps with the last four nucleotides of cap3.Generation of unintended mutations in either the cyclase and/or Nuc-SAVED could have significant consequences for the interpretation of cyclase activity and phage challenges assays reported anywhere the CBASSΔCap2ΔCap3 strain is used.
Lines 317-320, there is discussion that the lack of the native host context may be responsible for the absence of a clear phenotype for the PspA-cyclase conjugation.The use of the B. cereus CBASS "…expressed under a strong, constitutive B. subtilis promoter (PdegQ)…" (Lines 86 -87) might be masking a PspA phenotype.The discovery of the specific PspA-cyclase conjugate is a remarkable finding, demonstration of its relevance to the initiation and deployment of the CBASS in vivo is important to bolster the significance of this discovery.I would strongly suggest the authors generate two B. subtilis strains (pspA+ and pspA-) where the CBASS is regulated by the native promoter from B. cereus, rather than PdegQ.Evaluating the antiphage activity of these two strains against the four Groe phage in plaquing assay and planktonic challenges is likely the best chance to reveal a cyclase-PspA phenotype that would leave no doubt as to its relevance to bacterial immunity.

Secondary Comments:
Line 150, previously in Fig. 1 the cyclase single amino acid variant A331E alone lost all ability to prevent phage invasion in the population (presumably no longer a Cap2 substrate), I was surprised to see that this variant was never used again in lieu of the Cterminal-truncated cyclase in later experiments (e.g., Fig. 2).Was there a notable reason for using this more substantially mutated gene as opposed to the single point mutation?Substitution of the truncated C-terminal cyclase for the single A331E variant requires demonstration that the response to phage challenge is the same between the two cyclase mutants (e.g., Fig. 1, does the truncated cyclase also fail to prevent phage infection).
Lines 215-216, It is hard to evaluate if the Westerns in Extended Data Fig. 4

show that
Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work.The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.more cyclase is associated with the membrane fraction compared to the cytosolic fraction without a reference protein sample for relative intensity comparisons within and across blots (e.g., purified cyclase of a known concentration run alongside the two fractions to compare intensities)?Lines 323-328, please comment on how similar or divergent the Bce Cap2 is from the V. cholerae, E. coli, and P. aeruginosa enzymes previously tested.It seems that the Bce Cap2 studied here may have a unique mechanism to interact with PspA that is not present in these other Cap2 orthologs.Perhaps the utilization of PspA as a substrate could be predicted in other Cap2 enzymes from such a comparison?Line 326-330, Does the heterologous E. coli host not encode a native pspA, is the abundance of this native PspA too low to detect HMW cyclase conjugates, or is the native E. coli PspA an inferior Cap2 substrate?Also, now that the experiments are being performed in a new host, please explicitly state which pspA allele is being over-expressed in E. coli.Line 422-424, "…to prevent activation of CBASS in the absence of phage … CBASS uses its protein modification machinery to conjugate the cyclase to the highly conserved PspA protein…" I am unconvinced that conjugation of the cyclase to PspA is preventing spurious activation of this CBASS in the absence of phage.It appears the cyclase is in an inactive form in the absence of phage in a manner independent of its post-translational conjugation to PspA.This is evident in Fig. 2a where there is over-expression of the cyclase and constitutive expression of the Nuc-SAVED effector (i.e., massive amounts of un-conjugated cyclase and effector, but no killing).Additionally, the inactivity or limited activity of purified cyclase in Fig. 5f (conjugated and unconjugated) also suggests the cyclase is primarily activated by an unknown factor (as stated in Line 412).The presence or absence of PspA also does not appear to influence the ability of CBASS to effectively limit phage predation (Fig. 4c).Therefore, based on the data in Figs.4d,e and that mentioned above, it would be more appropriate to say the conjugation of the cyclase to PspA could be a means to slow cyclase activation during infection (rather than prevent activation in the absence of phage).
Additional Comments / Observations [These do not require responses; I only want to bring them to the authors' attention and are all marked with an *] *In general, the absence of asterisks for relevant p-values appears to be a systemic oversight throughout the manuscript (e.g., Fig. 1C and Fig. 4), including Extended Data Figs.
*In general, consistency across all figures with the usage of white and magenta triangles for identification of monomeric proteins and cyclase-PspA conjugates would be helpful.That being said, I found the most useful application of triangles was in Fig. 3a where only the magenta triangle was common across blots and included the name cyclase-PspA while the individual monomeric proteins were called out with arrows by name (rather than white triangles).This simplified and uniform identification scheme would greatly simplify interpretation of many of the blots and gels.*Line 98, Fig. 1c -Error bars are difficult to see, and it appears a formatting error occurred Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work.The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spectrometry."This statement should refer to Extended Data 9c rather than b.
Reviewer #2 (Remarks to the Author): The work from Krüger et al. describes the regulation of the CBASS system from B. cereus in B. subtilis.After a clear and up-to-date introduction, the authors demonstrate with strong proofs that the nucleotide cyclase is conjugated by the protein Cap2 to the ubiquitous membrane protein PspA.This sequestration of the cyclase to the membrane prevents non-relevant activation of the CBASS system.The protein Cap3 has been shown to be necessary for the deconjugation of the Cylclase from PspA.These findings present a real advancement in the understanding of the regulation of phage defense system in bacteria, and especially for CBASS.This work presents a strong interest for every research community with interests in phage defense systems.The paper is well written and made with solid science.The conclusions drawn by the authors are clear, careful and rely on clear proof and serious studies.The M&M, as well as the legends are nicely detailed.All experiments related to the main message of the articles are flawless and well made, but I noticed some points of the study or the discussion could be improved by some additional information/discussions.Overall, I'll recommend the publication of this article after a round of minor revision and say that the authors accomplished an incredible work.
Here are the main questions/critics/suggestions that I have regarding the manuscript: -Taxonomy of the four new phages L90-95 + Supp table 1: You did a great job to make taxonomic attributions to these new phages, unfortunately they are now obsoletes (see https://doi.org/10.1007/s00705-022-05694-2). I'll strongly suggest you to update their names and taxonomic distribution (forget myoviridae et al families and go deeper in classification if you can (see https://doi.org/10.3390/v9040070for an up-to-date guide)).I'll also recommend adding to Table S1 a column saying if the phage is virulent or temperate, since this information is important regarding the phenotype of lysis plaques with Geo23.
-L111-113: While it's really intriguing that CBASS does not provide the same resistance to B. subtilis, if the infection is made on agar or in liquid medium, I realized that you used two different temperatures for these tests (37°C and 30°C respectively).It'll be better to see the plate assay from Fig. 1 and Fig. S2 made at the same temperature for Geo23, as such parameter can significantly the outcome of the infection.
-It's really intriguing that infection by the phage Geo21 can be stopped by the CBASS system, since this phage belongs to the Jumbo-phage family.Some studies suggest that the phage genome is shielded early during the infection process, explaining its resistance against the CRISPR-Cas system (https://doi.org/10.1038/s41586-019-1786-y).The observation that the CBASS system is efficient even against Jumbo-phages suggest that the effector protein (Nuc-SAVED) is able to get in the shell-structure to degrade both phage and bacterial DNA.Confirming this fact can be done easily, and the information could really improve the impact of this study on our understanding of the CBASS system.But, since it's not about the main message of this study, I am not requiring it as a major review.Confirmation of degradation of phage/bacterial DNA in infected cells could be assessed with Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work.The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. a qPCR assay; or by microscopy with any DNA binding agent like DAPI.
-Figure 1: The difference in the sensitivity of the different phages to CBASS is really intriguing, and some elements bring by the authors (L113-123) may explain why Geo23 is poorly targeted by the CBASS system.An easy explanation for these differences could also be the eclipse period of each phage (time required to perform a lytic cycle after DNA injection.This paper and some others have clearly shown that CBASS is a system becoming active quite late during the infection process.I think it would be useful if the authors provide information about the eclipse periods of the different phages used in this study, in a way to rule out that Geo23 is almost insensitive to CBASS just because it lyses the bacteria faster than the CBASS system can kill it. -The model proposed in Fig. 6 and during the final discussion (L430-435) is nice and fit the paper data.Nonetheless, I am wondering if the authors could expand their model regarding the cyclase-Cap2 interaction.In Fig. 3a, it seems clear that Cap2-Cyclase complexes are "fused together", creating the multiples high molecular weight species observed on the gel.Do the authors think that this in vitro assay could be a clue that the cyclase is activated by oligomerization, of Cap2-cyclase complex first, and then by an unknown mechanism, Cap2 are removed from the cyclase oligomer.The need for high ATP concentration to form these complexes (Fig 3a .)and low ATP concentration for cyclase activation could be part of this mechanism too.I think more discussions are needed about this in the discussion.
-L212-216 and Fig. S4 : it is not crystal clear for me how the authors can make a comparison of the quantity of cyclase in the cytoplasm and along the membrane, since there are performing a pull-down for the cytoplasmic fraction, but not for the membrane one.I would like to see explanation about this comparison clarified somewhere.

Minor text modifications & typos:
General remark about the figures : the "*" representing p-values are missing in all figures of the paper.In the M&M, for IPTG induction, it is not always clear for how long and at which temperature the induction has been made, and if the culture were shacked.Also, some description of centrifuge speed in rpm, while g are more convenient for transposition to another model of centrifuge.L31-33 : The sentences sound weird, even if I get the main idea.Talking about p bacterial defense system for eukaryotic sound out of topic.L109 : please add « in our experimental conditions » L115 : Are any of these four phages close to each others?L116 : Author may add (if available) the identity of the gene coding for the shell-protein in Geo21's genome, and information about it in this paragraph.L121 : two "the" in the sentence -> the the indicator.L140-150 : Please explain why the cyclase could still be in the membrane fraction with its C-terminal deleted?Do you think the isopeptide bond is made with other proteins with other residue in the cyclase, as you showed L216-218?L261-263 : I think precision about the "lytic phenotype" should be made.Are you talking about its ability to bypass the CBASS resistance, or about the size/clarity of the lytic plaques.L224-225 : Please remind the reader that this binding has already been seen before in Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work.The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
other works, as you state in the introduction (ref 22,23).L230-231 : I disagree on this point, the light band/smear observed in the ΔCap3 & ΔpspA might suggest that these species exist, but in a lower quantity.Unless you have proteomics data to refute my point.L251-253 : And the proteomics data didn't reveal conjugation on other residues of the protein?Precision should be made about this in the text.L271-272 : Does the ΔpspA have any growth defect compared to a wild type?L665 : the word « Infection » does not belong to this sentence.L747 : I think that "his-tagged protein" should be replacer by "bacterial pellet" L746-750: the sentence about resuspension seems to be repeated.L750 : Could you please provide the parameters of the sonication, and bring precision if the sample was keept on ice or not.L1020-1021 : Left and right should be replace by top and bottom.
Reviewer #3 (Remarks to the Author): In the study, Krüger et al. explore mechanistic aspects of the CBASS anti phage defense system in Bacillus cereus.
The experiments are generally well designed and follow logic progression.Expressing the CBASS system in Bacillus subtilis and E. coli, they find that the CBASS component Cap2 conjugates the CBASS cyclase to the abundant protein PspA.They find that Cap3 is required to cleave the cyclase-PspA conjugation, to release the now active cyclase, which can then produce the cyclic nucleotide signal to launch the CBASS cascade.

General comments:
Overall, the manuscript appears rushed and is difficult to follow with many errors as exemplified below.
The methods section is particularly difficult to read.This distracts from the overall findings and makes it very difficult for the reader to follow.
The authors should rewrite the manuscript to better guide the reader.In its current form, the paper is targeted specifically for the CBASS field and not a broader microbiology audience.The introduction fails to properly describe the CBASS system, including the key role of the cyclic nucleotides.The authors do not mention that it is an abortive infection system.An introductory figure with an overview of the CBASS system would greatly improve the manuscript.
There are issues with the font in many of the figures, e.g.Fig. 4B.
The methodology is generally not explained sufficiently for others to be able to reproduce the data.The plasmid cleavage assay and the nucleotide extraction procedures are particularly unclear.
Several key terms are not defined: eg.CRISPR, ESCRT, Nuc-SAVED (not introduced until Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work.The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

line 381).
The nomenclature is inconsistent.Throughout the manuscript, standard nomenclature for naming genes and proteins are confused.E.g.

Specific comments:
The authors introduce four new phages.They rely on specific time points after infection to determine de effect on CBASS, but the timing of phage lifecycles is not determined.Therefore it is unclear at which point in the phage lifecycle the tests are performed.Fig .1.The significance values are not plotted.Fig. 4a.Why not show the free cyclase, to show that it gets decoupled and not e.g.degraded?Fig. 4d.and Extended Fig. 7b appear to follow a similar setup, but the non-infected controls are dissimilar and it is unclear from the figure legend why this would be.Fig. 4e.Why no SD?Fig. 6.The figure would benefit from adding more text in connection with the arrows.Line 23: "after infection by the isopeptidase Cap3".This sentence is confusing.Line 30: "The best-known examples for bacterial defence systems are the adaptive and innate immune system of eukaryotes".This is confusing.Line 44-46: The references are missing.Line 64: "To increase our understanding of CBASS regulation".This implies testing how the system is regulated in its native host.The statement should be reworded to better reflect the focus of the study, which is more mechanistic in nature.Line 87: The authors introduce the ∆6 strain.They explain that it lacks 5 prophages.What is the 6th deletion?Line 146: "A-coupled beads".A is not explained.Line 194: The names of strains are introduced.These names are not used consistently and seem out of place here.Line 263: "showed the most lytic phenotype (Fig. 1b), supposedly inducing the most drastic CBASS response and, thus, making it an ideal candidate to study our system in vivo".CBASS affects the Goe23 plaque phenotype but does not affect the PFU, so it is unclear why it is the ideal candidate.Why not choose one of the phages where CBASS reduces PFU? Line 263: "the most lytic phenotype".I suggest "most clear plaques".Line 286: "infection drop assay".I suggest "plaque assay".Line 288: It is not explained what cA3 is.Line 301: "This confirmed that the cyclase is inactive in the absence of phage".This is a very strong statement based on the data.The assay does not measure cyclase activity directly and relies on heterologous expression of the cyclase, followed by indirect measurement on the effector.Line 308: The authors refer to Fig. 1d, which is missing.Line 309: "Cap2 is essential for activation".Why was the cap2 mutant not tested in this assay?
Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work.The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
Line 322: This section seems out of place with the flow of the story.Line 436: The authors discuss whether PspA could sense initial phage injection-mediated membrane stress and activate the CBASS system, but in line 301 they conclude that the system senses phage infection around 30 min post infection, similar to what has been observed before.They should discuss this in relation to their comment on PspA.Line 438: "survival program".CBASS is an abortive infection suicide program, not a survival program.

Author Rebuttal to Initial comments
We are grateful to the reviewers who provided extensive, constructive and helpful comments for our manuscript.We have addressed every point made by the three reviewers below.In the revised manuscript we include a substantial amount of additional experimental data to meet the expectations of the reviewers.Specifically these include: • Repetition of conjugation assays with PspA from Bacillus cereus o This new data confirms that Cap2 is able to use both PspA homologs (PspA Bsu and PspA Bce ) as substrates for cyclase conjugation.We have to note, however, that purification of the PspA Bce protein was challenging and the protein is not very stable.In addition, we identified conjugation target residues on PspA Bce .This new data is discussed in the revised version of the manuscript and can be found in Extended Data o These data provide evidence that the catalytic Cap2 mutants and the deletion mutant behave in the same way regarding their ability to activate the cyclase (Fig. 4d, Extended Data Fig. 7) and provide immunity (Fig. 1b, c).

• Analysis of cyclase activity in response to infection with different phages by nucleotide
extraction of infected cultures and effector activation assays (Fig. 4e, f, g) o We extracted nucleotides over a broader range of time after infection with phage Goe23 and studied Nuc-SAVED activation in the plasmid cleavage assay.This revealed a significant increase of plasmid cleavage activity with nucleotides extracted 25 minutes post infection in the pspA deletion strain compared to the CBASS wild type (Fig. 4e, Extended Data Fig. 9).Repeating this experiment with phage Goe21 and Goe26, however, did not show significant differences between the CBASS and CBASS ΔpspA strains.These observations are discussed and put in context.
Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work.The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.o This showed that low expression of CBASS strongly reduces immunity in B. subtilis against infection with phage Goe21, Goe23, and Goe26.Interestingly, deletion of pspA in such cells provided some protection against phage Goe21 and Goe26 in liquid culture.This new data can be found in Fig. 4c and Extended Data Fig. 6c and supports the idea that conjugation to PspA does affect immunity and phenotypes may be masked in the overexpressed system.fine) would greatly improve the significance of these findings.

Malcolm
I want to commend the authors for their hard work, and I implore them to please read the following comments with the knowledge that they are meant to be helpful and written with a voice of curiosity and encouragement.There is no malice intended by any language that follows and in no way are they meant to be combative, rude, or demeaning.It is likely that some of these ideas and observations presented may reflect my own misinterpretation of data or text, despite my best efforts to be thorough.Please know that I respect your time, effort, and diligence in preparing this manuscript and thank you for reporting these interesting results.
Response: We thank the reviewer for the kind words about our work.

Primary Comments:
The characteristics of both plaquing and planktonic growth of B. subtilis Δ6 + CBASS challenged with Goe23 are dramatically different from the other phages tested (Figs. 1B & 1C,Ext Data Fig 2).The Goe23 plaquing phenotype and the crash in OD (~3-5h) could be indications of slow activation of CBASS that is specific to this phage, as these are not seen in similar challenges with the three other phage.Because the delayed time to activation (~ 30 min) of this CBASS is an important conclusion of this study, it is important to demonstrate this is not a phenomenon specific to Goe23 infection alone (Figs. 4b,d,e and Ext.Figs. 6 and 7) (i.e., is delayed Cap3 deconjugation/activation of the cyclase only happening with Goe23).Additional temporal experiments (Similar to those performed in Ext.Figs. 6 and 7) should be performed using at least one additional phage.Perhaps Goe26 might be the most reasonable follow-up phage to use as both planktonic and plaquing data are currently presented, which could ease the burden of these experiments.
Response: We thank the reviewer for the suggestions to include other phages.The revised version of the manuscript contains additional effector activation experiments with nucleotides extracted from CBASSexpressing cells infected with Goe21 and Goe26 (Fig. 4f, g, supporting previous Ext.Fig. 7).We extracted nucleotides from cells infected with phage Goe21, Goe23 or Goe26 after different time points post infection and measured their ability to activate the cognate CBASS effector Nuc-SAVED in a plasmid cleavage assay.These experiments confirm that the cyclase is activated after infection with all three phages.Interestingly, we detected CBASS nucleotides only 25 min, 35 min and 45 min after infection with phage Goe23, Goe26 and Goe21, respectively.This late activation of CBASS during the infection cycle is in agreement with our previous observations.We also observed a significant increase of plasmid cleavage activity with nucleotides extracted 25 minutes post infection with phage Goe23 in the pspA deletion strain compared to the CBASS wild type (Fig. 4e, Extended Data Fig. 9).
Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work.The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.Lines 253-254, "The low sequence conservation between PspA homologs may explain why Cap2 requires flexibility in target recognition (Extended Data Fig. 5c)".The in vitro assay is a neat tool and I commend the authors for the monumental task of purifying all these components and establishing a condition that recapitulates some of the aspects observed in their in vivo conditions.However, the in vitro condition likely has some substantial artifactual activity where the Cap2 enzyme appears more promiscuous in its conjugation of the cyclase to alternative substrates/residues (e.g., high MW complexes of Cap2-cyclase in Fig. 3a not seen in vivo, Figs.2a, 2d, Extended Data 3a, Extended Data Fig. 9a).However, in vivo results demonstrate cyclase-specific peptides are found only on residues K147 and K220 (Fig. 2b, Lines 176-177, Lines 332-333).To suggest that there is flexibility in target recognition by Cap2 in vivo activity to facilitate its ability to work with diverse PspA substrates (i.e., function in diverse bacterial hosts), the authors need to perform similar purifications and Western blots as those presented in Fig. 2d using strains containing the K147R and K220R variants of the B. subtilis PspA and demonstrate whether comparable alternative Lys residues on PspA are truly utilized as substrates in vivo.It is also important to assess the capacity of Cap2 to conjugate the cyclase to B. cereus PspA, which is presumably the "natural" substrate, and determine the locations of preferred Lys residues for cyclase conjugation.
Response: We realize that the determination of the target lysine residue on PspA is limited by several points.Firstly, as mentioned by the reviewer, we do not know the importance of the two lysine residue in vivo.And secondly, we likely miss modified lysine residues in our MS analysis, due to the generation of peptides unsuitable for detection.To address these points, we analysed the conjugation of the cyclase to B. cereus Psp A in our in vitro conjugation assay.Although the Bce homolog was not very stable, we observed conjugation of the cyclase to PspA Bce .This shows that Cap2 is capable of using both PspA homologs as substrates for conjugation.This new data can be found in Extended Data Fig. 5d.
In Fig. 4d, use of either of the Cap2 inactive Cys->Arg variants (Figs.1b,c, 5a) is important to demonstrate the utility of conjugation and would be more convincing than the double CBASSΔCap2ΔCap3 currently presented (i.e., the variants are a precision tool for addressing the role of conjugation while the double mutant is a hammer).If it is impractical to repeat these experiments with the cap2 variants, it is important to justify the use of the double knockout mutant in lieu of these superior strains (this is also applicable to Fig. 2d, but more critical to address in Fig. 4d where cyclase activity is explored in vivo).
Response: We thank the reviewer for pointing out this issue.We repeated the experiment with the catalytic mutants of Cap2 and this data can be found in the revised version of the manuscript.Plasmid cleavage cannot be observed with both mutants, same as the Cap2-Cap3 double deletion mutant, indicating that the cyclase is not activated in such cells.On another note, we added the Cap2-Cap3 Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work.The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.double deletion mutant to the infection assays of Fig. 1b,c to complement both observations.In conjunction with the previous comments, would the authors please double check that in generating the double cap2 cap3 deletion strain (e.g., Fig. 4d) an unintended catalytically inactive chimeric cyclase has not been created (i.e., the native cyclase stop codon is preserved) and the Nuc-SAVED effector is still likely to be functional (i.e., start codon is preserved, a putative ribosome binding site exists, and transcription of the effector is unlikely to be interrupted.. basically check for any obvious indications of polar effects on the effector).There is overlap between the final nucleotide of the cyclase stop codon and the first nucleotide of the cap2 start codon and the Nuc-SAVED effector overlaps with the last four nucleotides of cap3.Generation of unintended mutations in either the cyclase and/or Nuc-SAVED could have significant consequences for the interpretation of cyclase activity and phage challenges assays reported anywhere the CBASSΔCap2ΔCap3 strain is used.
Response: We agree that it is essential to confirm that the sequence of any of the constructs used is flawless.As described in the supplementary table, the double cap2 cap3 mutant has been generated as follows: amplification of the cyclase gene, cloning into the vector pGP1460, amplification of the effector gene with a forward primer adding a RBS, and cloning into the vector downstream of the cyclase gene (pLK04).The plasmid has been sequenced and we can confirm that both genes are complete from start to stop.
Lines 317-320, there is discussion that the lack of the native host context may be responsible for the absence of a clear phenotype for the PspA-cyclase conjugation.The use of the B. cereus CBASS "…expressed under a strong, constitutive B. subtilis promoter (PdegQ)…" (Lines 86 -87) might be masking a PspA phenotype.The discovery of the specific PspA-cyclase conjugate is a remarkable finding, demonstration of its relevance to the initiation and deployment of the CBASS in vivo is important to bolster the significance of this discovery.I would strongly suggest the authors generate two B. subtilis strains (pspA+ and pspA-) where the CBASS is regulated by the native promoter from B. cereus, rather than PdegQ.Evaluating the antiphage activity of these two strains against the four Groe phage in plaquing assay and planktonic challenges is likely the best chance to reveal a cyclase-PspA phenotype that would leave no doubt as to its relevance to bacterial immunity.
Response: We agree that the overexpression of CBASS could mask the regulatory function of the cyclase conjugation.To address this issue, we mutated the hyperactive PdegQH promoter (very strong) back to the wild type PdegQ promoter with very low activity (as demonstrated by Stanley 2005).With such low expression, CBASS was unable to confirm notable immunity against any of the phages tested in this study in a plaque assay (Extended Data Fig. 6c).Interestingly, when we compared the initial response to phage infection in liquid medium, we found that in the absence of PspA, low CBASS expression provided Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work.The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.some protection against phage Goe21 and Goe26, but not Goe23 (Fig. 4c).This indicates that under low CBASS expression, presence of PspA is inhibitory for CBASS immunity, likely due to conjugation of the majority of the cyclase to PspA.These new data can be found in Fig. 4c and Ext.Fig. 6c.

Secondary Comments:
Line 150, previously in Fig. 1 the cyclase single amino acid variant A331E alone lost all ability to prevent phage invasion in the population (presumably no longer a Cap2 substrate), I was surprised to see that this variant was never used again in lieu of the C-terminal-truncated cyclase in later experiments (e.g., Fig. 2).Was there a notable reason for using this more substantially mutated gene as opposed to the single point mutation?Substitution of the truncated C-terminal cyclase for the single A331E variant requires demonstration that the response to phage challenge is the same between the two cyclase mutants (e.g., Fig. 1, does the truncated cyclase also fail to prevent phage infection).
Response: The bulk of the work presented was carried out with the truncated variant, both in vitro and in vivo, before the A331E variant was tested, so the reason for this was essentially technical.We confirmed that the truncated cyclase had a very similar basal cyclase activity to the wild-type protein, so we are reasonably confident that the two variants are functionally comparable.Due to the requirement for extensive further experimentation to address the key points raised by reviewers we did not carry out further tests to compare the two "tail" variants.
Lines 215-216, It is hard to evaluate if the Westerns in Extended Data Fig. 4 show that more cyclase is associated with the membrane fraction compared to the cytosolic fraction without a reference protein sample for relative intensity comparisons within and across blots (e.g., purified cyclase of a known concentration run alongside the two fractions to compare intensities)?
Response: We agree and removed the statement about quantitative distribution from the text.Lines 323-328, please comment on how similar or divergent the Bce Cap2 is from the V. cholerae, E. coli, and P. aeruginosa enzymes previously tested.It seems that the Bce Cap2 studied here may have a unique mechanism to interact with PspA that is not present in these other Cap2 orthologs.Perhaps the utilization of PspA as a substrate could be predicted in other Cap2 enzymes from such a comparison?Response: We thank the reviewer for bringing up this interesting point.We compared the Bce Cap2 protein with the homologs and the sequence conservation is indeed not as high.The Vibrio and E. coli proteins share 51% sequence identity (72% similarity), while the Bce protein shares only 15-17% Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work.The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.sequence identity (35-36% similarity) with either of them.This is not entirely unexpected as Bacillus cereus is more distant in the tree.It would be interesting to investigate the interaction between Cap2 and PspA further to understand whether interacting residues in Cap2 are conserved.We added the following sentence to the manuscript: "The Bce Cap2 protein is phylogenetically more distant and shares only around 35% amino acid sequence similarity with these homologs.".Line 326-330, Does the heterologous E. coli host not encode a native pspA, is the abundance of this native PspA too low to detect HMW cyclase conjugates, or is the native E. coli PspA an inferior Cap2 substrate?Also, now that the experiments are being performed in a new host, please explicitly state which pspA allele is being over-expressed in E. coli.
Response: Even though E. coli and B. subtilis both encode for PspA homologs, the context and the Psp system work very differently.In E.coli, PspA forms an inhibitory complex with PspF, thereby preventing induction of the Psp response.This seems to make PspA unavailable as a substrate of Cap2.In B. subtilis on the other hand, PspA is not associated with another protein of the Psp system in uninducing conditions.We have added a clarifying sentence to our discussion.We also explicitly state now which pspA allele is overexpressed in E. coli.Line 422-424, "…to prevent activation of CBASS in the absence of phage … CBASS uses its protein modification machinery to conjugate the cyclase to the highly conserved PspA protein…" I am unconvinced that conjugation of the cyclase to PspA is preventing spurious activation of this CBASS in the absence of phage.It appears the cyclase is in an inactive form in the absence of phage in a manner independent of its post-translational conjugation to PspA.This is evident in Fig. 2a where there is overexpression of the cyclase and constitutive expression of the Nuc-SAVED effector (i.e., massive amounts of un-conjugated cyclase and effector, but no killing).Additionally, the inactivity or limited activity of purified cyclase in Fig. 5f (conjugated and unconjugated) also suggests the cyclase is primarily activated by an unknown factor (as stated in Line 412).The presence or absence of PspA also does not appear to influence the ability of CBASS to effectively limit phage predation (Fig. 4c).Therefore, based on the data in Figs.4d,e and that mentioned above, it would be more appropriate to say the conjugation of the cyclase to PspA could be a means to slow cyclase activation during infection (rather than prevent activation in the absence of phage).
Response: We agree and have reworded the relevant text as suggested.
Additional Comments / Observations [These do not require responses; I only want to bring them to the authors' attention and are all marked Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work.The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
find.Additionally, more information is required in the figure legend to understand the experiments depicted in the gels.(e.g., the use of R1 and R2 in the first-row of the gel legends, "30" in the secondrow, and the third-row numerals 1, 2, 5, 15, and 30).Response: This has been changed.

Reviewer #2 (Remarks to the Author):
The work from Krüger et al. describes the regulation of the CBASS system from B. cereus in B. subtilis.After a clear and up-to-date introduction, the authors demonstrate with strong proofs that the nucleotide cyclase is conjugated by the protein Cap2 to the ubiquitous membrane protein PspA.This sequestration of the cyclase to the membrane prevents non-relevant activation of the CBASS system.The protein Cap3 has been shown to be necessary for the deconjugation of the Cylclase from PspA.These findings present a real advancement in the understanding of the regulation of phage defense system in bacteria, and especially for CBASS.This work presents a strong interest for every research community with interests in phage defense systems.The paper is well written and made with solid science.The conclusions drawn by the authors are clear, careful and rely on clear proof and serious studies.The M&M, as well as the legends are nicely detailed.All experiments related to the main message of the articles are flawless and well made, but I noticed some points of the study or the discussion could be improved by some additional information/discussions.Overall, I'll recommend the publication of this article after a round of minor revision and say that the authors accomplished an incredible work.
Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work.The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.not about the main message of this study, I am not requiring it as a major review.Confirmation of degradation of phage/bacterial DNA in infected cells could be assessed with a qPCR assay; or by microscopy with any DNA binding agent like DAPI.
Response: We agree that the ability of CBASS to provide resistance against a jumbo phage is interesting and provides opportunity to understand the mechanism of CBASS further.It is quite possible that degradation of the host chromosome, outside the phage shell, is sufficient to confer immunity.We have now added some discussion of that point, but we feel that further experimentation is beyond the scope of the current paper -Figure 1: The difference in the sensitivity of the different phages to CBASS is really intriguing, and some elements bring by the authors (L113-123) may explain why Geo23 is poorly targeted by the CBASS system.An easy explanation for these differences could also be the eclipse period of each phage (time required to perform a lytic cycle after DNA injection.This paper and some others have clearly shown that CBASS is a system becoming active quite late during the infection process.I think it would be useful if the authors provide information about the eclipse periods of the different phages used in this study, in a way to rule out that Geo23 is almost insensitive to CBASS just because it lyses the bacteria faster than the CBASS system can kill it. Response: We thank the reviewer for raising this interesting point.A similar issue has been addressed by reviewer 1.In the revised manuscript, we include new experimental data on the activation of the cyclase after infection with different phages (Fig. 4 e, f, g).We extracted nucleotides from cells infected with phage Goe21, Goe23 or Goe26 after different time points post infection and measured their ability to activate the cognate CBASS effector Nuc-SAVED in a plasmid cleavage assay.These experiments confirm that the cyclase is activated after infection with all three phages.Interestingly, we detected CBASS nucleotides only 25 min, 35 min and 45 min after infection with phage Goe23, Goe26 and Goe21, respectively.Comparing this to the infection kinetics of wild type bacteria (named "no CBASS" in the figure) shown in Extended Data Fig. 2, it provides further evidence that CBASS is activated late in the infection cycle.At the moment we do not have enough experimental evidence to confidently argue whether Goe23 is only minorly affected by CBASS because of its short infection cycle, or because it encodes anti-defence proteins.
-The model proposed in Fig. 6 and during the final discussion (L430-435) is nice and fit the paper data.Nonetheless, I am wondering if the authors could expand their model regarding the cyclase-Cap2 interaction.In Fig. 3a, it seems clear that Cap2-Cyclase complexes are "fused together", creating the multiples high molecular weight species observed on the gel.Do the authors think that this in vitro assay could be a clue that the cyclase is activated by oligomerization, of Cap2-cyclase complex first, and then Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work.The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.by an unknown mechanism, Cap2 are removed from the cyclase oligomer.The need for high ATP concentration to form these complexes (Fig 3a .)and low ATP concentration for cyclase activation could be part of this mechanism too.I think more discussions are needed about this in the discussion.
Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work.The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.identifier for the different MW bands being discussed (e.g., a-d) Lines 401-417, the cyclase product of unknown ATP composition which elutes around 3 minutes is very intriguing -being most prominent in the 5 mM ATP condition.Upon seeing this again, in the revised manuscript, I'm too tempted to whisper in the authors' ears that this could be a very interesting molecule to pursue (in a different study, of course!).Could this represent an inhibitor of Cap3 activity or an antagonist of CA3 activation of Nuc-SAVED?Identification of this molecule and MS quantification of its abundance pre-and post-infection or inclusion in the established in vitro Cap3 deconjugation and plasmid cleavage assays might reveal some hidden role for this curious nucleotide product.
Extended Data Figures 3a-b, these gels look to have the "LC" labeled on different bands Extended Data Figure 3 Legend Title, "conjugates" to "conjugated" Reference 20, an updated version of this biorxiv has just been published in a peer-reviewed journal, PMID: 38172623 (I am not an author of this study) Line 747-748 (Method: Phage infection assays), "…600 nm (OD600) was adjusted to 1.0 and the cells were used to inoculate a 96 well plate…" Is there a further dilution from OD660 of 1.0 that is performed?All liquid phage challenges appear to start around 0.1 OD600.
Reviewer #2: Remarks to the Author: In my opinion Krüger et al. did a great job during this revision round.They perfectly answered the critics and comments made by me, but also by the other referees.The additional experiments that they performed in the last months really helped.The overall quality of the manuscript clearly increased and I think it is now ready to be published in this journal.
Reviewer #3: Remarks to the Author: Krüger et al. have made a great effort in addressing the comments and suggestions and they have made extensive revisions to their manuscript.In particular, key concepts are now introduced to a broader audience, and the addition of the native promotor data nicely captures some of the nuances of how effective the CBASS system may be in connection with PspA, in the native Bacillus cereus strain.I still highly recommend that the authors measure the timing of the phage life cycles/ latent period, as also suggested by another reviewer.This would allow the reader to compare the timing of CBASS activation in this system, compared to the first reports on CBASS.
Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work.The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
Additionally, I have the following minor comments: Title: Swapping the word "regulates" for "activate" or "trigger" would better reflect the conclusions.The final sentence of the abstract is unclear.There are still nomenclature issues, e.g.lines 342 and 1082 (Escherichia coli).Line 46: The sentence implies that CRISPR-Cas is exclusively activated by viral DNA/RNA.This statement should be reworded to reflect that this is one of many mechanisms that activate CRISPR-Cas.Ref. 13 is not appropriate for this statement.Lines 473-474.This statement is very bold and is not supported by the data since the experiments are not carried out in the native system under non-induced conditions.I suggest deleting this statement.
Reviewer #4: Remarks to the Author: Krüger et al. have made a great effort in addressing the comments and suggestions and they have made extensive revisions to their manuscript.In particular, key concepts are now introduced to a broader audience, and the addition of the native promotor data nicely captures some of the nuances of how effective the CBASS system may be in connection with PspA, in the native Bacillus cereus strain.I still highly recommend that the authors measure the timing of the phage life cycles/ latent period, as also suggested by another reviewer.This would allow the reader to compare the timing of CBASS activation in this system, compared to the first reports on CBASS.
Additionally, I have the following minor comments: Title: Swapping the word "regulates" for "activate" or "trigger" would better reflect the conclusions.The final sentence of the abstract is unclear.There are still nomenclature issues, e.g.lines 342 and 1082 (Escherichia coli).Line 46: The sentence implies that CRISPR-Cas is exclusively activated by viral DNA/RNA.This statement should be reworded to reflect that this is one of many mechanisms that activate CRISPR-Cas.Ref. 13 is not appropriate for this statement.Lines 473-474.This statement is very bold and is not supported by the data since the experiments are not carried out in the native system under non-induced conditions.I suggest deleting this statement.
Reviewer #5: Remarks to the Author: Krüger et al. have made a great effort in addressing the comments and suggestions and they have made extensive revisions to their manuscript.In particular, key concepts are now introduced to a broader audience, and the addition of the native promotor data nicely captures some of the nuances of how effective the CBASS system may be in connection with PspA, in the native Bacillus cereus strain.I still highly recommend that the authors measure the timing of the phage life cycles/ Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work.The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.latent period, as also suggested by another reviewer.This would allow the reader to compare the timing of CBASS activation in this system, compared to the first reports on CBASS.
Additionally, I have the following minor comments: Title: Swapping the word "regulates" for "activate" or "trigger" would better reflect the conclusions.The final sentence of the abstract is unclear.There are still nomenclature issues, e.g.lines 342 and 1082 (Escherichia coli).Line 46: The sentence implies that CRISPR-Cas is exclusively activated by viral DNA/RNA.This statement should be reworded to reflect that this is one of many mechanisms that activate CRISPR-Cas.Ref. 13 is not appropriate for this statement.Lines 473-474.This statement is very bold and is not supported by the data since the experiments are not carried out in the native system under non-induced conditions.I suggest deleting this statement.

Final Decision Letter:
Message: 7th March 2024 Dear Malcolm, I am pleased to accept your Article "Reversible conjugation of a CBASS nucleotide cyclase regulates bacterial immune response to phage infection" for publication in Nature Microbiology.Thank you for having chosen to submit your work to us and many congratulations.
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Fig. 5c.• Addition of Cap2 catalytic mutants to infection assays (Fig 1b, c) and effector activation assays with extracts from CBASS uninfected and infected cells (Fig. 4d, Ext.Data Fig. 7) In this study Krüger et al. describe the reversible conjugation of a CD-NTase (cyclase) to the ubiquitous phage shock protein (PspA) and propose a model by which this conjugation plays a role in the sequestration of the cyclase to limit spurious activation of the Type II CBASS system in the absence of phage infection.They first show the genetic and catalytic requirements of the B. cereus CBASS for protection against phage predation in the heterologous B. subtilis host and further show the unexpected finding that this cyclase is conjugated specifically to the B.subtilis PspA protein in a Cap2 dependent manner.The Cap3 enzyme is also shown to scarlessly proteolyze the cyclase-PspA conjugate in vitro and in vivo, and this activity occurs late during infection with phage Groe23.The cyclase in vitro appears to be in a state of reduced activity, regardless of its conjugated state, in vitro and inactive in vivo in the absence of phage.This suggests that a critical condition to activate the cyclase during infection remains to be discovered, and the authors suggest based on in vitro cyclase assays this could be related to the abundance of intracellular nucleotides (e.g., ATP).Heroic efforts were made to demonstrate the utility of the PspA-cyclase conjugate in vivo, but the physiological role of this interaction remains enigmatic, with no conclusive evidence demonstrating this interaction between the host PspA and the CBASS cyclase is necessary for defense against phage by the Type-II CBASS.The expansion of these findings to include additional phage infection experiments (e.g., Groe26) validating the delayed activation hypothesis, demonstration of cyclase conjugation to diverse PspA substrates (not only B. subtilits PspA), as well as testing the CBASS antiphage activity under control of its native promoter (B.subtilis host is Open Access This file is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.In the cases where the authors are anonymous, such as is the case for the reports of anonymous peer reviewers, author attribution should be to 'Anonymous Referee' followed by a clear attribution to the source work.The images or other third party material in this file are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material.If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
Response: Extended Data Fig. 8 is now referred to in the main body of the text.Descriptions have also been added to the figure legend.*Line 1066, Extended Data Figure 9a, please number the band boxes on the gel that were analyzed by MS.Response: The boxes have been numbered.*Line 1083, "…conjugate cleavage assay from b was analysed by intact mass spectrometry."This statement should refer to Extended Data 9c rather than b.