A glycine zipper motif governs translocation of type VI secretion toxic effectors across the cytoplasmic membrane of target cells

  1. Jemal Ali
  2. Manda Yu
  3. Li-Kang Sung
  4. Yee-Wai Cheung
  5. Erh-Min Lai

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Abstract

Type VI secretion systems (T6SSs) can deliver diverse toxic effectors into eukaryotic and bacterial cells. Although much is known about the regulation and assembly of T6SS, the translocation mechanism of effectors into the periplasm and/or cytoplasm of target cells remains elusive. Here we use the Agrobacterium tumefaciens DNase effector Tde1 to unravel the mechanism of translocation from attacker to prey. We demonstrate that Tde1 binds to its adaptor Tap1 through the N-terminus, which harbours continuous copies of GxxxG motifs resembling the glycine zipper structure found in proteins involved in the membrane channel formation. Amino acid substitutions on G 39 xxxG 43 motif does not affect Tde1-Tap1 interaction and secretion but abolish its membrane permeability and translocation of its fluorescent fusion protein into prey cells. The data suggest that G 39 xxxG 43 governs the delivery of Tde1 into target cells by permeabilizing the cytoplasmic membrane. Considering the widespread presence of GxxxG motifs in bacterial effectors and pore-forming toxins, we propose that glycine zipper mediated permeabilization is a conserved mechanism used by bacterial effectors for translocation across target cell membranes.

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  1. Version published to 10.1101/2022.07.12.499750v2 on bioRxiv
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    Manuscript number: RC-2022-01588

    Corresponding author(s): Erh-Min, LAI

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    1. General Statements [optional]

    This section is optional. Insert here any general statements you wish to make about the goal of the study or about the reviews.

    The authors thank the reviewers for the positive and valuable comments, which have helped us to improve the quality of this work. We have addressed all comments by providing additional data and/or explanation with a detailed point-by-point response. The revised manuscript included new data: 1) viable cell counts of growth inhibition assay (Fig. 2A), 2) Quantitative data of microscope data (Fig. 2C, Fig. 4), 3) quantitative data of interabacterial competition (Fig. 5A, 5B), western blotting data of growth inhibition (Fig. S1A and S1B), secretion assay of single glycine-zipper mutants (Fig. 5C), and inclusion of full gel of western blot results (Fig. S3 and S5). By integrating these new results, we have substantially strengthened the findings that a glycine zipper motif of a type VI secretion effector T6SS Tde1contributes to its translocation across the cytoplasmic membrane of target cells.

    2. Point-by-point description of the revisions

    This section is mandatory. *Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. *

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    Summary: In this manuscript, Ali et al. propose that a glycine zipper motif located at the N-terminus of the Agrobacterium tumefaciens T6SS DNase effector, Tde1, can transport the toxin across the cytoplasmic membrane and into the cytoplasm, where its target is found. To support these claims, they perform a series of secretion, competition, toxicity, and fluorescence microscopy assays showing that a mutation in two glycine residues affects toxicity of the effector during competition and its ability to enter a target cell, but not its secretion through the T6SS or its binding to the adaptor protein Tap1. The concept brought forth in this study is quite interesting and important - the notion that T6SS effectors have domains that aid in their transport into the cytoplasm of the target cell. This is similar to a recent finding that a domain common to bacterial pyocins and T6SS effectors can mediate DNase toxin transport through the target cell's cytoplasmic membrane (Atanaskovic et al., mBio, 2022); the authors should mention and discuss this recent work. Nevertheless, it is my impression that the results do not fully support the conclusions and proposed mechanism, even though the general idea seems correct.

    Ans: We thank this reviewer found this work interesting and important. We hope the revised manuscript including the new data and careful interpretation have substantialized the conclusions and proposed mechanisms. We also included the excellent work by Atanaskovic et al., 2022 and discussed the findings in the revision (see lines 344-349).

    Major comments:

    • An experiment that directly demonstrates the ability of the glycine zipper to mediate transport of a toxin across a membrane would greatly support and solidify the conclusions of this work. For example, showing the ability of a purified protein to enter spheroplasts or liposomes in a glycine zipper dependent fashion. Currently, the authors perform experiments that can only indirectly support the proposed function of the glycine zipper to enable the effector to cross the membrane, and as detailed below, some of these experiments are over-interpreted in my opinion. Ans: We agree that the direct evidence for the ability of the glycine zipper to mediate Tde1 transport across target cell membrane is to perform the *in vitro *translocation assay. Unfortunately, the attempts to purify sufficeint amounts of full-length or N-termial version of Tde1 have not been successful. Therefore, we are unable to perform this experiment. Accoringly, we have tried our best to carefully interpret the data and rephrase the statements accordingly.

    • Lines 153-159: It is not clear how much these results are relevant to the activity of the glycine zipper motif during effector delivery by the T6SS. If I understand correctly, the described experiments are of over-expression of the proteins in the E. coli cytoplasm, where glycine zipper-dependent membrane permeability and toxicity are detected. However, one would expect that if the effector is to be transported from the periplasm to the cytoplasm during T6SS delivery, then the glycine zipper should function from the periplasmic face of the cytoplasmic membrane, and not from its cytoplasmic face, as is the case in these experiments. Is it possible that the observed toxicity and membrane permeability be the result of over-expression in the "wrong place"? Ans: The reviewer is right that Tde1 should permealize cytoplasmic membrane from periplasmic side upon injection from the attacker based on our proposed model. The purpose of ectopic expression of Tde1 and its variant in *E. coli *is to dissect the region and motif of Tde1 DNase-independent toxicity and the ability in enhancing membrane permeability regardless of which sides of cytoplasmic membrane the Tde1 mediates toxicity and permeability. The results of glycine zipper-dependent toxicity and membrane permeability provide a ground work for the experiments of secretion and interabacterial compeittion in the context of active T6SS action to determine the role of glycine zipper in Tde1 export and translocation.

    • Fig. 4B: This figure appears to be very important, and the authors base a large part of their main conclusion regarding the role of the glycine zipper in membrane crossing on it. However, some controls are missing and part of the results observed in the figure do not match their description in the text. • Lines 233-237 - While the authors state in the text that GFP and mCherry signals did not overlap in E. coli cells co-cultured with Agrobacterium cells expressing Tde1(M)-GLGL, I see many double-colored cells in this sample (bottom panels in Fig. 4B). Actually, all cells appear to have both green and blue colors, except for a few cells that are only green but that also seem to be dead judging by their ghostly appearance in the phase contrast channel.

    Ans: We thank the reviewer pointed this out. By looking at this particular image more carefully, it is striking that the majority of cells seem to emit both green and blue colors from this Tde1(M)GLGL sample. We have performed a total of three indepenent experiments for this translocation assay and all results except this particular sample in this particular experiment are consistent in all three independent experiments. Honestly, we could not explain this result and a possibility is this sample might be accidentally mixed with another sample. Because this is the only sample with inconsistent result with another two independent experiments, we decided NOT to use the results from this independent experiment and instead performed another independent experiment. We now have included the quantitative data from three effective independent experiments and show the representative images in Figure 4.

    How is it that all cells in the bottom panels are blue (indicating that they are E. coli target cells)? Shouldn't a large portion of the cells be Agrobacterium cells that should not be blue, since these are added at the beginning of the competition assay at a 10:1 ratio in their favor? Ans: As explained above, we have no defined answer and decided to perform additional repeats, which are consistent with results of another two independent experiments.

    It is quite remarkable that so much GFP signal is transported into the E. coli target cells so that it is so clearly visible under the microscope. How do the authors know that the GFP signal overlapping with the mCherry is really inside the cell and not outside (for example, proteins secreted to the media that attach to the cell envelope)? Will the GFP signal remain if trypsin is added to the media before visualization under the microscope? Ans: Indeed, our quantitative data show there are ~50% cells have GFP overlapping with mCherry in the translocation positive samples. The signals should be inside the cells because no overlay signals were observed from N-Tde1GLGL or Tde1(M)GLGLeven though they are secreted.

    Can the authors quantify the ratio of E. coli cells that have overlapping green and blue colors over several experiments for each sample, to show that this phenomenon repeats and is statistically significant? Ans: Yes, see quantitative data in Figure 4.

    Can the authors explain why at least some of these E. coli cells should not be dead due to the toxicity mediated by the third effector of the Agrobacterium T6SS, Tae? Ans: In *Agrobacterium tumefaciens *C58, Tde1/2 are the major effectors contributing to antibacterial activity. Tae effector has little impact on interbacterial competition outcome (see previous publications Ma et al., 2014 doi: 10.1016/j.chom.2014.06.002.; Yu et al., 2020 doi.org/10.1128/JB.00490-20)

    Why were the microscopy competitions performed differently than the regular competition assays? Why wasn't AK media used in these competitions? How active is the T6SS under these conditions compared to the AK media? Ans: We have tried to use AK medium for the translocation assay but only very weak fluorescent signals can be observed likely due to the low expression when grown on this nutrient poor medium. In order to correlate the results of the compeittion assay with translcoation experiment, we have performed *E. coli *killing assay using LB medium that is used for translocation experiment now. For the interbacterial competition against agrobacterial siblings, we still used AK medium for competition because no detectable interbacterial compettion activity could be observed between two *A. tumefaciens *strains on LB agar. As reported earlier, stronger interbacterial competition outcome was detected from co-culture on AK than other nutrient rich medium while the secretion activity grown in AK medium is lower (Yu et al., 2020 doi.org/10.1128/JB.00490-20). These results indicate that the factors other than secretion activity also impacted recipient cell susceptibity, which however is not the main focous of this work.

    In the N-Tde1 sample, many Agrobacterium cells appear to have the GFP signal in foci rather than distributed throughout the cell (as it is in other samples), while the E. coli cells have a uniform and strong GFP signal. Can the authors comment on that? Ans: Thanks the reviewer for raising this question.We are also curious about the Tde1 glycine zipper-dependent GFP foci and now include this potential explanation in the Discussion of revised manuscript (line 387-406). To this end, we do not have an answer for it. Because glycine zipper repeats are known to interact with membrane, it is possible that Tde1 proteins may preferntially bind to microdomain of cytoplasmic membrane, which was recently found in A. tumefaciens (Czolkoss et al., 2021). We also found that Tde1 proteins (either tagged with HA or GFP) are proned for truncation when they are ectopically expressed in *E. coli *or when Tdi1 is absent or not equivalent. Thus, it is possible that Tde1-GFP proteins are truncated after translocation into *E. coli *cells, in which most GFP signals are emitted from free GFP instead of Tde1-GFP. The stability of free GFP derived from translocated Tde1-GFP may also explain the high percentage of *E. coli *cells exhibiting overlayed GFP/mCherry signals.

    It might be easier for readers to visualize this figure and see the signal distribution in the different cells if the authors show a zoomed in version in the main text, and provide the wide field images as a supplementary figure. Ans: We have tried to include zoom-in images but the resolution is not good. We have improved the quality of images in the Figure 4 and believe the images are clear to see individual and overlayed fluorescence signals.

    • Fig. 5C-D: The reduced expression and secretion of the GLGL mutant is considerable. How can the authors rule out that this reduction was the cause for the reduced observed toxicity of the mutant in 5A-B? Moreover, the results show that the GLGL double mutant is hampered in expression, secretion, and DNase activity, and it negatively affects overall T6SS activity. Since this mutant was used throughout the paper, and in the absence of a direct assay showing membrane transport mediated by the glycine zipper motif, the claim of the role of this motif in membrane crossing is not well substantiated by the results. If the authors were to show that the single glycine mutants used in Fig. 5D, which are stable and have an intact DNase activity, behave as claimed in the final conclusion sentence (lines 279-283), then the conclusions will be better substantiated by the results. Ans: Thank you very much for suggesting this important experiment. We have now constructed the single G39L and G43L variants expressed together with Tdi1 in *A. tumefaciens tdei *mutant for both secretion and interbacterial competition assays (see description in lines 259-280 and Fig. 5). As shown in Figure 5, both G39L and G43L variants are expressed and secreted at similar or even higher levels than wild type Tde1 but have no detectable antibacterial activity against either *E. coli *or *A. tumefaciens *1D1609. This result substantializes the role of this glycine zipper motif in translocation.

    Minor comments:

    • Line 93: I am not sure that Ntox15 should still be referred to as a "novel" domain.

    Ans: despite the evidence of this domain as DNase, the name of Ntox15 is used. We think to keep this nomenclatture as it will be easier to be ditinquished from other nuclease or toxin domain.

    • Line 105: The section's heading does not actually describe its content. The results here only show toxicity upon over-expression of the effector or its mutant forms in E. coli. Therefore, this cannot be referred to as a "prey cell" since the effector was not transported into it during competition. Moreover, the results in Fig. 5A do not support DNase-independent toxicity during competition. Ans: The heading is changed to “Tde1 exhibits DNase-independent growth inhibition in* E. coli*” (line 115).
    • Please consider making all of the symbols in the growth assays semi-transparent. It is impossible to discern between the different, overlapping curves. Ans: The growth curve results are improved by changing line colors and reducing size bars (Fig. 1B, 1C; Fig. 2A, 2D)
    • Please consider making the size bars in all microscopy images more pronounced. They are barely visible in their current form. Also, it would be better to show images of the same magnification/zoom for the different samples, since the current presentation shows cells from different samples at different sizes, and it can be confusing to the readers. Ans: Amended (Fig. 2C; Fig. 4).
    • In Fig. 1B and in Fig. 2A the authors show that expression of Tde1(M) in cells is toxic, yet in Fig. 2D they see no toxicity. Can the authors please comment on this discrepancy? Ans: Fig. 2D showed the viability of *E. coli *cells after Tde1 variants were induced for 1 hr before ONPG uptake assay to indicate the increased membrane permeability is not due to cell death. In Fig. 1B, the growth inhibition of Tde1(M) is also not evident at 1 hr. So, the results are consistent.
    • I am not convinced that the assay in Fig. 2E can be used to determine bacteriostatic/bacteriolytic effect. It is not clear how such a distinction can be made from OD measurements, since an increase in OD can result from the entire population growing after removal of the stressor, or just part of the population that did not lyse/die. To make such a claim, the authors can spot bacteria on repressing media at different timepoints after protein induction, and then determine CFU.

    Ans: The increased OD600 value during recovery could be caused by either resumed cell division or cell elongation. Based on the newly added growth inhibition assay of all Tde1 variants which we showed nice correlation between CFU counting and OD600value (Fig. 2A, S2) and no increased cell size/length of *E. coli *cells expressing N-Tde1 or Tde1(M), we think the recovered OD600 value is supportive of N-Tde1 or Tde1(M) exhibiting bacteriostatic toxicity. In addition to that, our interbacterial competition data showed that Tde1(M)-Tdi1 which is still having intact glycine zipper doesn’t show significant detectable killing, supporting the bacteriostatic function of Tde1 glycine zippers. In fact, we performed this experiment based on Mariano et al.(Nat. Commun. 2019 doi: 10.1038/s41467-019-13439-0), which showed the recovery of OD600 value after removal of inducer as the evidence that the Ssp6 toxin is not bacteriolytic.

    • Fig. 3A: A control is missing. To verify that the N-terminal part of Tde1 is not promiscuously interacting with proteins, the authors should include a control sample testing its inability to precipitate a protein other than Tap1 in the same experiment. Ans: Our previous study has showed that Tde1 can co-immunprecipiate Tap1 but not a non-T6SS protein RpoA (Bondage et al., 2016 doi:10.1073/pnas.1600428113), indicating that Tde1 is not promiscuously interacting with proteins. Considering the tight biochemical interaction between Tap1 with N-Tde1 but not C-Tde1 that correlate with their ability for secretion upon loading onto VgrG1, N-Tde1 is unlikely to bind proteins non-specifically. This is also supported by the non-specific protein bands from cellular fractions recognized by anti-Tap1 are not co-immunoprecipitated by any of Tde1 variants (Fig. S3). We could repeat the experiments to include additional proteins as negative controls but we chose to use time for other more critical experiments during the limited revision time.

    • Fig. 3B: the blots are very "dirty". It is not clear how the authors were able to determine expression and precipitation of some truncations (for example, C2-Tde1 in the E. coli IP panel looks like a background band found in other lanes too).

    Ans: We agree that western blots of co-IP experiments in *E. coli *are not very clear due to the weak signals of some Tde1 variants and background. As pointed out by the reviewer 3, this result is not conclusive and rovide little additional information other than the co-IP results from *A. tumefaciens. *Because the interaction between Tde1 variants and Tap1 when expressed in *E. coli *are not physiologically relevant and not the main focus of this work, we have removed the E. coli co-IP results from this manuscript as suggested by the reviewer 3.

    • Lines 222-225 (Fig. 4A): I can't see C-1-Tde1(M)-sfGFP in the cellular blot. All the bands in this lane look like background bands that are also present in all other lanes. Therefore, I am not sure how the conclusion regarding this truncation's ability to be secreted was reached. Ans: We agree that C1-Tde1(M)-sfGFP is barely detectable due to its weak signal overlapping with cross-reacted bands. Since several attemps to improve the western blot quality by changing antibody and pre-blocking with protein lysages of vector control strain did not produce convincing results for detection of C1-Tde1(M)-sfGFP, we have rephrased the description of this result as “However, C1-Tde1(M)-sfGFP protein signal could not be unambiguously determined in the cellular fraction due to the overlapping of its predicted protein band with cross-reacted proteins, and no corresponding C1-Tde1(M)-sfGFP band was detected in the extracellular fraction.” (line 234-237).

    • Fig. 4A: the protein names above the lanes should include the sfGFP that is fused to them. Ans: Amended.

    • It would be preferable to show quantitative competition assays with statistics rather than pictures of a plate showing a single competition result, if conclusions or observations on minor differences in toxicity are made (for example, line 253: "The killing activity of Δtdei(Tde1GLGL-Tdi1) was largely compromised"). Since the authors performed each competition assay more than once, these data should be available to them. Ans: Amended. We have repeated the interbacterial competition experiments including single G39L and G43L variants for multiple biological repeats (see detailed in legends of Fig. 5A, 5B). The quantitative data with statistical analysis were added, which show no statistical difference of any glycine zipper mutants as comapred to Tde1(M) or when expressed in the T6SS mutant. Thus, there are no detectable antibacterial activity of glycine zipper mutants against either *E. coli *or *A. tumefaciens *siblings.
    • Fig. 5A: The author claim at the beginning of the manuscript (first results section heading: "Tde1 can cause DNase-independent growth inhibition of prey cells") that the N-terminal region of Tde1 is toxic on its own in the prey cell, yet in this competition assay Tdi1(M) shows no toxicity against the E. coli target cells. In the microscopy assay (Fig. 4B), it appears that a lot of Tdi1(M) enters the prey cell, since we can visualize it under the microscope. Can the authors clarify this discrepancy and explain why they do not expect to see target killing by this mutant even though they claimed it is toxic earlier? Ans: As describbed in earlier response, N-Tde1 amd Tde1(M) toxicity can exhibit toxicity by ectopic expression in *E. coli. *We mainly used this ectopic expression assay to dissect the region and motif contributing the toxicity. Compared to the interbacterial competiton process where Tde1(M) may only transiently permealze cytoplasmic membrane transiently as the final destination is cytoplasm where wild type Tde1 but not Tde1(M) exerts DNase toxicity. Thus, the toxicity of N-Tde1 and Tde1(M) can be only observed when the proteins are continuously produced in the cytoplasm. The role of N-Tde1, specifically the glycine zipper motifs, is to mediate Tde1 translocation across inner membrane, instead of exerting toxicity during the context of interbacterial competition.
    • Fig. 5B: the GLGL mutant seems to have some residual toxicity, not dissimilar to what is shown in 5A. Why are these similar results interpreted differently (in 5A they are "largely compromised", while in 5B "killing activity... was not detectable")? Also, why was Tde(M)1-Tdi1 used in Fig. 5A but Tdi1(M) without the immunity gene used in Fig. 5B? Ans: As described above, to better quantify the interbacterial competition outcomes, we have repeated the interbacterial competition experiments and used Tde(M)1-Tdi1 instead of Tdi1(M) for at least three biological replicates. The quantitative data with statistical analysis were carried out to clarify this ambiguity (Fig. 5A, 5B).
    • Fig. 5: Does the remaining third effector, Tae, not play a role in these competition assays? If, as shown in Fig. 5C, the entire T6SS is less active when a GLGL mutant is expressed, couldn't the different in toxicity shown in Figs. 5A-B be the result of lack of Tae secretion and toxicity?

    Ans: As decribed above, Tae effector has little impact on interbacterial competition outcome. The quantatitive interbacterial competition results (Fig. 5A, 5B) also clarify the ambiguity because single G39L and G43L variants are expressed and secreted at similar or even higher levels than wild type Tde1 but have no detectable antibacterial activity against either *E. coli *or *A. tumefaciens *1D1609.

    • Lines 359-362: T6SS effectors that bind the inner Hcp tube were suggested to be only partially folded. Ans: Amended.

    Reviewer #1 (Significance (Required)):

    The concept of T6SS effectors providing their own mechanism of transport from the cytoplasm to the periplasm is very interesting. It will appeal to audience in a wide range of microbiology disciplines, including those interested in toxins, membrane transport, and even translational applications. A similar concept was recently proposed and demonstrated for a domain that is also found in T6SS effectors (Atanaskovic et al., mBio, 2022).

    Expertise: I have been studying the different aspects of T6SS for the past decade.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    This manuscript is focused on understanding how the Agrobacterium tumefaciens T6SS effector, Tde1, is translocated across the cell envelope of target cells and how this effector binds to the adapter Tap1. The authors show that GxxxG motifs in the N terminal region of Tde1 are required for delivery into the cytoplasm of target cells and permeabilising the cytoplasmic membrane. Given that these GxxxG motifs resemble glycine zipper structures that are found in proteins involved in membrane channel formation, the authors propose that these Tde1 motifs are involved in channel formation in the target cell. The authors also show that the N terminal region of Tde1 binds to Tap1 to facilitate loading onto the T6SS machinery but that the GxxxG motifs are not involved in this binding. Overall the manuscript was easy to read and followed a logical presentation of the findings. There are a few major comments that this reviewer has below - addressing these would allow the authors' claims to be more robustly supported. Ans: Thank you very much for the positive comments and valuable suggestion. We hope the revised manuscript including the new data and careful interpretation have substantialized the conclusions and proposed mechanisms.

    Major comments:

    1. Fig 1B: Why is this such a short growth experiment (5 hrs total with 2 hr pre and 3 hrs post induction)? Reporting on a growth experiment would normally be at least until the cells reach stationary phase but here the cells are still clearly in exponential phase. This reviewer would query what happens to growth rate in later exponential growth and into stationary phase? Is the toxic effect lessened in later stages of growth? Ans: We have indeed performed the growth curve analysis with longer time period. However, we noted that the growth at later time points are not always consistent and our interpretation is that the continuous expression of toxins may lead to the selection of mutants. Since the 3 or 4 hr time period already showed the toxicity phenotype, we have focused on this time frame for the growh experiments.

    2. It is indeed surprising that C2-Tde1(WT) does not inhibit growth despite it having a functional DNase domain and being expressed in the cytoplasm. Did the authors confirm that this protein variant was expressed by Western blot or other means? This should be done to confirm that this variant is indeed not impacting upon growth instead of it not impacting growth simply because it is not being expressed.

    Ans: Amended. All Tde1 variants including C2-Tde1 are expressed (data included in Fig S1)

    1. The letters used to report significance are not clear to this reviewer. The authors say that "The significant differences were shown by the different letters (p value

    For all fluorescence microscopy experiments how many fields of view were imaged for each biological replicate? Were the fields selected at random or was the field selection biased to what was present in the field before taking the image? The answers to all of these questions should be stated in the methods. Also the microscopy data presented in the manuscript is not quantitative. Quantification of the number of cells with PI vs Hoechst signal (in Fig 2C) and mcherry vs gfp signal (in Fig 4B) for all fields of view and for all biological replicates would be very informative and convince the reader that the authors have not just "cherry picked" the images they are showing in the manuscript. This could be performed manually or the authors could use the freely available image analysis program Fiji (https://imagej.net/software/fiji/) to perform these analysis in a semi-automated manner.

    Ans: The number of images and experiments were now described in the figure legends and the quantititive data are included (Fig. 2C).

    1. For the co-IP experiments in Fig 3 where interaction between HA tagged Tde1 and Tap1 is demonstrated the authors should also show that Tap1 does not interact with a different HA-tagged protein i.e. that the interaction is specific to Tde1 and not the HA motif. Ans: All Tde1 variants were tagged with HA. As shown in Fig. 3A, Tap1was not co-precipitated by C2-Tde1 and C1-Tde1(M), indicating that Tap1 specifically interacts with N-terminal region of Tde1.

    For all Western blot images there should be at least 2 protein standard markers present in each individual blot - i.e. for Fig 3A and B the bottom panel showing Tap1 detection only has the 35 kDa marker, it should have at least one more marker in it. The same is true for other panels in Fig 2, 3 and 4. Having at least two molecular weight markers in a panel is now standard for most journals when presenting Western blot images. Ans: Amended. We have now included the full gel of western blot results in Fig. S3 and S5 of those shown in main figures.

    For the competition assay serial dilution images in Fig 5A-B the images are a nice way to visually represent the experimental outcome but they should accompany graphs showing the competitive index of CFU/ml of the input prey and attacker vs the output prey and attacker for all biological replicates. This will convince the reader that the authors had equivalent amounts of the prey and the attacker going into the experiment and also that all attackers grew at the same rate and so were equally able to target the prey cell. This quantification could also provide more convincing out competition of ID1609 prey by C58 attacker (Fig 5B). Ans: Amended. As indicated above, we have repeated the interbacterial competition experiments for at leaset three biological replicates and show that quantitative data with statistical analysis (Fig. 5A, 5B).

    Minor comments:

    Line 40: should read "...demonstrate that the effector itself..." Ans: The sentence has been rephrased (line 40) .

    Line 41: "...we propose..." instead of "...we proposed..." since present tense makes more sense for this statement.

    Ans: Amended (line 42).

    Line 51: "Each specialized protein secretion system" instead of "Each of...." Ans: Amended (line 52).

    Line 76: "A glycine zipper structure..."

    Ans: Amended (line 83).

    Line 79: "For example..."

    Ans: Amended (line 86).

    Lines 96-100: The present tense should be used here as the current usage of past tense implies that this has been done in previous work and not in the current study - eg "we revealed", "we showed" would be better as "we reveal", "we show".

    Ans: Thanks for the advice. We have made changes throughout the manuscript.

    Fig 5B - The competition assay serial dilution images look a bit blurry, are there images the authors could use that are not blurry?

    Ans: Amended. As indicated above, we now show quantitative data with statistical analysis (Fig. 5A, 5B).

    Reviewer #2 (Significance (Required)):

    This work is significant in as while there is a great deal known about how T6SS effectors cause toxicity there is less known about how these effectors are loaded onto the T6SS machinery and very little known about how T6SS effectors are able to translocate across the cytoplasmic membrane of target cells to reach a cellular component that is in the cytoplasm. This work would be of wide general interest to researchers in the T6SS field as well as those interested in bacterial secretion systems.

    Reviewer expertise key words: Molecular microbiology, T6SS, interbacterial competition

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    EVIDENCE, REPRODUCIBILITY AND CLARITY

    Summary:

    In this work, Ali et al. demonstrate that the N-terminal GxxxG motif of the T6SS DNase effector Tde1 of Agrobacterium tumefaciens is required for interbacterial intoxication. Using a combination of cell viability, reporter, and microscopy assays, the authors demonstrate that over-expression of the N-terminus of Tde1 results in inner membrane permeability. Moreover, the authors show that both the interaction between Tde1 and its adaptor Tap1 as well as the T6SS-mediated secretion of Tde1 are dependent on the N-terminus of Tde1. Finally, using a combination of in vitro and in vivo experiments, the authors determine that the N-terminal GxxxG motif is essential to Tde1-dependent interbacterial killing by enabling effector entry into competing bacterial cells.

    Major comments:

    If N-tde1 is 1-97 aa, the predicted size is 9 kDa, but it shows up as ~17 kDa? Can the authors comment on this? Does N-tde1 or tde1 dimerize? Ans: The theoretical Mw of N-Tde1-HA is 10.64 KDa, which indeed migrated at higer position ~17 kDa. It is notable that full-length Tde1 with theoretical 29.5-kDa migrated slower in SDS-PAGE with a observed size ~36 kDa as observed previously (Ma et al., 2014 doi:10.1016/j.chom.2014.06.002). Similarly, the full-length HA-tagged Tde1(M) with theoretical 30.89 kDa migrated at a position ~38 kDa. Since the protein samples analyzed by SDS-PAGE including reducing agent, we cannot exclude the possibility that Tde1 or N-Tde1 may form dimer or oligomer that was disrupted by SDS-PAGE but it appears not forming dimer on SDS-PAGE.

    I have many concerns with the data and conclusions drawn from the data in Fig. 3B. I recommend removing it since (1) the data are not accurately represented in the text and (2) it is difficult to ascertain whether biologically relevant conclusions can be drawn from what happens with Agrobacterium proteins in E. coli. Below is a summary of my concerns regarding this section: I disagree with the authors' statements in lines 191-198. Their pulldown with E. coli is not consistent with their pulldown in C58. In fact, given the expression problems of some of the constructs in E. coli, I believe the data shown in Fig. 3B is inconclusive. The amount of Tap1 that co-IP'ed with N-Tde1GLGL and Tde1(M) is very low even though the expression levels of N-Tde1GLGL and Tde1(M) were relatively strong. Therefore, I do not feel confident concluding that these proteins "interact". Secondly, Tde1(M)GLGL was not expressed in E. coli, so no conclusions can be drawn. Moreover, the C1 and C2 variants were also not expressed well, so I believe the authors' statement in line 191-192: "Similar to the results in A. tumefaciens, the N-Tde1 and Tde1(M) interacted with Tap1 but not the C-terminal variants", is unjustified. You cannot rule out that C1 and C2 do not interact with Tap1 because C1 and C2, like Tde1(M)GLGL, were not expressed well in E. coli. Ans: We agree with the reviewer that the *E. coli *co-IP result is not conclusive due to the low expression and instability of proteins mostly during the process of cell lysis and purification, and it provides little additional information other than data from co-IP in *A. tumefaciens. *Because the interaction between Tde1 variants and Tap1 when expressed in *E. coli *are not physiologically relevant and not the main focus of this work, we have removed the E. coli co-IP results from this manuscript.

    Lines 211-214: It looks like C1-Tde1(M) inhibits T6SS secretion. I am aware that in Agrobacterium, it has been shown that effector loading is essential for secretion, but then why does the pTrc200 secrete Hcp? Also, in Fig. 4B, a strain expressing C1-Tde1(M) now secretes Hcp. Ans: Thanks for noting our previous finding that Tde loading is critical for secretion. Our data are indeed supportive of the effector loading in activating T6SS as only very low levels of Hcp secretion could be detected from the strain containing vector only or C1-Tde1(M). In our previous paper (Wu et al., 2020 https://doi.org/10.15252/embr.201947961), there is either little or no detection of Hcp secretions when effectors are not loaded, indicating that effector loading is important but not essential for Hcp secretion. Because overexpression of VgrG can also activate T6SS secretion in the absence of effector loading (Bondage et al., 2016 doi:10.1073/pnas.1600428113), we think the low level secretion under certain conditions could be caused by some cells with higher levels of VgrG protein concentration but more work is required to elucidate the underlying mechanisms.

    Minor comments:

    Fig. 2B could benefit from better labeling to indicate that most strains lack lacY. Also, why is BW25113 WT showing such a low OD420 if it has LacY? Or is WT without lacZ? Please clarify.

    Ans: We apologize for not labeling clearly. The BW25113 strain lacks lacZ, therefore all the ∆lacY strains were complemented with a plasmid encoding lacZ (pYTA-lacZ). We have now added the labels to avoid confusion (Fig. 2B).

    Reviewer #3 (Significance (Required)):

    SIGNIFICANCE

    It has been known for over a decade that T6SS effectors have both periplasmic and cytosolic targets (e.g., cell wall and DNA). However, it remains unclear (1) where within the target cell are T6SS effectors are delivered and (2) once delivered, how do effectors reach their intracellular target site. In this work, Ali et al. demonstrate that for Tde1, the N-terminal GxxxG motif is essential for Tde1 to reach its target (DNA). The authors identified Tde1 homologs in several bacteria, suggesting that this model may be relevant across a wide range of bacteria. Additional research is needed to (1) determine whether Tde1 is originally secreted into the periplasm and (2) understand how non-Tde1/non-GxxxG effectors reach their target site.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    In this work, Ali et al. demonstrate that the N-terminal GxxxG motif of the T6SS DNase effector Tde1 of Agrobacterium tumefaciens is required for interbacterial intoxication. Using a combination of cell viability, reporter, and microscopy assays, the authors demonstrate that over-expression of the N-terminus of Tde1 results in inner membrane permeability. Moreover, the authors show that both the interaction between Tde1 and its adaptor Tap1 as well as the T6SS-mediated secretion of Tde1 are dependent on the N-terminus of Tde1. Finally, using a combination of in vitro and in vivo experiments, the authors determine that the N-terminal GxxxG motif is essential to Tde1-dependent interbacterial killing by enabling effector entry into competing bacterial cells.

    Major comments:

    If N-tde1 is 1-97 aa, the predicted size is 9 kDa, but it shows up as ~17 kDa? Can the authors comment on this? Does N-tde1 or tde1 dimerize?

    I have many concerns with the data and conclusions drawn from the data in Fig. 3B. I recommend removing it since (1) the data are not accurately represented in the text and (2) it is difficult to ascertain whether biologically relevant conclusions can be drawn from what happens with Agrobacterium proteins in E. coli. Below is a summary of my concerns regarding this section: I disagree with the authors' statements in lines 191-198. Their pulldown with E. coli is not consistent with their pulldown in C58. In fact, given the expression problems of some of the constructs in E. coli, I believe the data shown in Fig. 3B is inconclusive. The amount of Tap1 that co-IP'ed with N-Tde1GLGL and Tde1(M) is very low even though the expression levels of N-Tde1GLGL and Tde1(M) were relatively strong. Therefore, I do not feel confident concluding that these proteins "interact". Secondly, Tde1(M)GLGL was not expressed in E. coli, so no conclusions can be drawn. Moreover, the C1 and C2 variants were also not expressed well, so I believe the authors' statement in line 191-192: "Similar to the results in A. tumefaciens, the N-Tde1 and Tde1(M) interacted with Tap1 but not the C-terminal variants", is unjustified. You cannot rule out that C1 and C2 do not interact with Tap1 because C1 and C2, like Tde1(M)GLGL, were not expressed well in E. coli.

    Lines 211-214: It looks like C1-Tde1(M) inhibits T6SS secretion. I am aware that in Agrobacterium, it has been shown that effector loading is essential for secretion, but then why does the pTrc200 secrete Hcp? Also, in Fig. 4B, a strain expressing C1-Tde1(M) now secretes Hcp.

    Minor comments:

    Fig. 2B could benefit from better labeling to indicate that most strains lack lacY. Also, why is BW25113 WT showing such a low OD420 if it has LacY? Or is WT without lacZ? Please clarify.

    Significance

    It has been known for over a decade that T6SS effectors have both periplasmic and cytosolic targets (e.g., cell wall and DNA). However, it remains unclear (1) where within the target cell are T6SS effectors are delivered and (2) once delivered, how do effectors reach their intracellular target site. In this work, Ali et al. demonstrate that for Tde1, the N-terminal GxxxG motif is essential for Tde1 to reach its target (DNA). The authors identified Tde1 homologs in several bacteria, suggesting that this model may be relevant across a wide range of bacteria. Additional research is needed to (1) determine whether Tde1 is originally secreted into the periplasm and (2) understand how non-Tde1/non-GxxxG effectors reach their target site.

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    Referee #2

    Evidence, reproducibility and clarity

    This manuscript is focused on understanding how the Agrobacterium tumefaciens T6SS effector, Tde1, is translocated across the cell envelope of target cells and how this effector binds to the adapter Tap1. The authors show that GxxxG motifs in the N terminal region of Tde1 are required for delivery into the cytoplasm of target cells and permeabilising the cytoplasmic membrane. Given that these GxxxG motifs resemble glycine zipper structures that are found in proteins involved in membrane channel formation, the authors propose that these Tde1 motifs are involved in channel formation in the target cell. The authors also show that the N terminal region of Tde1 binds to Tap1 to facilitate loading onto the T6SS machinery but that the GxxxG motifs are not involved in this binding. Overall the manuscript was easy to read and followed a logical presentation of the findings. There are a few major comments that this reviewer has below - addressing these would allow the authors' claims to be more robustly supported.

    Major comments:

    1. Fig 1B: Why is this such a short growth experiment (5 hrs total with 2 hr pre and 3 hrs post induction)? Reporting on a growth experiment would normally be at least until the cells reach stationary phase but here the cells are still clearly in exponential phase. This reviewer would query what happens to growth rate in later exponential growth and into stationary phase? Is the toxic effect lessened in later stages of growth?
    2. It is indeed surprising that C2-Tde1(WT) does not inhibit growth despite it having a functional DNase domain and being expressed in the cytoplasm. Did the authors confirm that this protein variant was expressed by Western blot or other means? This should be done to confirm that this variant is indeed not impacting upon growth instead of it not impacting growth simply because it is not being expressed.
    3. The letters used to report significance are not clear to this reviewer. The authors say that "The significant differences were shown by the different letters (p value <0.01)" and then have a, b, c etc next to lines of the growth experiments in Figure 1B, C and Fig 2A, B, E etc. Which comparisons have a p value <0.01? this is not clear.
    4. For all fluorescence microscopy experiments how many fields of view were imaged for each biological replicate? Were the fields selected at random or was the field selection biased to what was present in the field before taking the image? The answers to all of these questions should be stated in the methods. Also the microscopy data presented in the manuscript is not quantitative. Quantification of the number of cells with PI vs Hoechst signal (in Fig 2C) and mcherry vs gfp signal (in Fig 4B) for all fields of view and for all biological replicates would be very informative and convince the reader that the authors have not just "cherry picked" the images they are showing in the manuscript. This could be performed manually or the authors could use the freely available image analysis program Fiji (https://imagej.net/software/fiji/) to perform these analysis in a semi-automated manner.
    5. For the co-IP experiments in Fig 3 where interaction between HA tagged Tde1 and Tap1 is demonstrated the authors should also show that Tap1 does not interact with a different HA-tagged protein i.e. that the interaction is specific to Tde1 and not the HA motif.
    6. For all Western blot images there should be at least 2 protein standard markers present in each individual blot - i.e. for Fig 3A and B the bottom panel showing Tap1 detection only has the 35 kDa marker, it should have at least one more marker in it. The same is true for other panels in Fig 2, 3 and 4. Having at least two molecular weight markers in a panel is now standard for most journals when presenting Western blot images.
    7. For the competition assay serial dilution images in Fig 5A-B the images are a nice way to visually represent the experimental outcome but they should accompany graphs showing the competitive index of CFU/ml of the input prey and attacker vs the output prey and attacker for all biological replicates. This will convince the reader that the authors had equivalent amounts of the prey and the attacker going into the experiment and also that all attackers grew at the same rate and so were equally able to target the prey cell. This quantification could also provide more convincing out competition of ID1609 prey by C58 attacker (Fig 5B).

    Minor comments:

    Line 40: should read "...demonstrate that the effector itself..."

    Line 41: "...we propose..." instead of "...we proposed..." since present tense makes more sense for this statement.

    Line 51: "Each specialized protein secretion system" instead of "Each of...."

    Line 76: "A glycine zipper structure..."

    Line 79: "For example..."

    Lines 96-100: The present tense should be used here as the current usage of past tense implies that this has been done in previous work and not in the current study - eg "we revealed", "we showed" would be better as "we reveal", "we show".

    Fig 5B - The competition assay serial dilution images look a bit blurry, are there images the authors could use that are not blurry?

    Significance

    This work is significant in as while there is a great deal known about how T6SS effectors cause toxicity there is less known about how these effectors are loaded onto the T6SS machinery and very little known about how T6SS effectors are able to translocate across the cytoplasmic membrane of target cells to reach a cellular component that is in the cytoplasm. This work would be of wide general interest to researchers in the T6SS field as well as those interested in bacterial secretion systems.

    Reviewer expertise key words: Molecular microbiology, T6SS, interbacterial competition

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    Referee #1

    Evidence, reproducibility and clarity

    Summary:

    In this manuscript, Ali et al. propose that a glycine zipper motif located at the N-terminus of the Agrobacterium tumefaciens T6SS DNase effector, Tde1, can transport the toxin across the cytoplasmic membrane and into the cytoplasm, where its target is found. To support these claims, they perform a series of secretion, competition, toxicity, and fluorescence microscopy assays showing that a mutation in two glycine residues affects toxicity of the effector during competition and its ability to enter a target cell, but not its secretion through the T6SS or its binding to the adaptor protein Tap1. The concept brought forth in this study is quite interesting and important - the notion that T6SS effectors have domains that aid in their transport into the cytoplasm of the target cell. This is similar to a recent finding that a domain common to bacterial pyocins and T6SS effectors can mediate DNase toxin transport through the target cell's cytoplasmic membrane (Atanaskovic et al., mBio, 2022); the authors should mention and discuss this recent work. Nevertheless, it is my impression that the results do not fully support the conclusions and proposed mechanism, even though the general idea seems correct.

    Major comments:

    • An experiment that directly demonstrates the ability of the glycine zipper to mediate transport of a toxin across a membrane would greatly support and solidify the conclusions of this work. For example, showing the ability of a purified protein to enter spheroplasts or liposomes in a glycine zipper dependent fashion. Currently, the authors perform experiments that can only indirectly support the proposed function of the glycine zipper to enable the effector to cross the membrane, and as detailed below, some of these experiments are over-interpreted in my opinion.
    • Lines 153-159: It is not clear how much these results are relevant to the activity of the glycine zipper motif during effector delivery by the T6SS. If I understand correctly, the described experiments are of over-expression of the proteins in the E. coli cytoplasm, where glycine zipper-dependent membrane permeability and toxicity are detected. However, one would expect that if the effector is to be transported from the periplasm to the cytoplasm during T6SS delivery, then the glycine zipper should function from the periplasmic face of the cytoplasmic membrane, and not from its cytoplasmic face, as is the case in these experiments. Is it possible that the observed toxicity and membrane permeability be the result of over-expression in the "wrong place"?
    • Fig. 4B: This figure appears to be very important, and the authors base a large part of their main conclusion regarding the role of the glycine zipper in membrane crossing on it. However, some controls are missing and part of the results observed in the figure do not match their description in the text.
      • Lines 233-237 - While the authors state in the text that GFP and mCherry signals did not overlap in E. coli cells co-cultured with Agrobacterium cells expressing Tde1(M)-GLGL, I see many double-colored cells in this sample (bottom panels in Fig. 4B). Actually, all cells appear to have both green and blue colors, except for a few cells that are only green but that also seem to be dead judging by their ghostly appearance in the phase contrast channel.
      • How is it that all cells in the bottom panels are blue (indicating that they are E. coli target cells)? Shouldn't a large portion of the cells be Agrobacterium cells that should not be blue, since these are added at the beginning of the competition assay at a 10:1 ratio in their favor?
      • It is quite remarkable that so much GFP signal is transported into the E. coli target cells so that it is so clearly visible under the microscope. How do the authors know that the GFP signal overlapping with the mCherry is really inside the cell and not outside (for example, proteins secreted to the media that attach to the cell envelope)? Will the GFP signal remain if trypsin is added to the media before visualization under the microscope?
      • Can the authors quantify the ratio of E. coli cells that have overlapping green and blue colors over several experiments for each sample, to show that this phenomenon repeats and is statistically significant?
      • Can the authors explain why at least some of these E. coli cells should not be dead due to the toxicity mediated by the third effector of the Agrobacterium T6SS, Tae?
      • Why were the microscopy competitions performed differently than the regular competition assays? Why wasn't AK media used in these competitions? How active is the T6SS under these conditions compared to the AK media?
      • In the N-Tde1 sample, many Agrobacterium cells appear to have the GFP signal in foci rather than distributed throughout the cell (as it is in other samples), while the E. coli cells have a uniform and strong GFP signal. Can the authors comment on that?
      • It might be easier for readers to visualize this figure and see the signal distribution in the different cells if the authors show a zoomed in version in the main text, and provide the wide field images as a supplementary figure.
    • Fig. 5C-D: The reduced expression and secretion of the GLGL mutant is considerable. How can the authors rule out that this reduction was the cause for the reduced observed toxicity of the mutant in 5A-B? Moreover, the results show that the GLGL double mutant is hampered in expression, secretion, and DNase activity, and it negatively affects overall T6SS activity. Since this mutant was used throughout the paper, and in the absence of a direct assay showing membrane transport mediated by the glycine zipper motif, the claim of the role of this motif in membrane crossing is not well substantiated by the results. If the authors were to show that the single glycine mutants used in Fig. 5D, which are stable and have an intact DNase activity, behave as claimed in the final conclusion sentence (lines 279-283), then the conclusions will be better substantiated by the results.

    Minor comments:

    • Line 93: I am not sure that Ntox15 should still be referred to as a "novel" domain.
    • Line 105: The section's heading does not actually describe its content. The results here only show toxicity upon over-expression of the effector or its mutant forms in E. coli. Therefore, this cannot be referred to as a "prey cell" since the effector was not transported into it during competition. Moreover, the results in Fig. 5A do not support DNase-independent toxicity during competition.
    • Please consider making all of the symbols in the growth assays semi-transparent. It is impossible to discern between the different, overlapping curves.
    • Please consider making the size bars in all microscopy images more pronounced. They are barely visible in their current form. Also, it would be better to show images of the same magnification/zoom for the different samples, since the current presentation shows cells from different samples at different sizes, and it can be confusing to the readers.
    • In Fig. 1B and in Fig. 2A the authors show that expression of Tde1(M) in cells is toxic, yet in Fig. 2D they see no toxicity. Can the authors please comment on this discrepancy?
    • I am not convinced that the assay in Fig. 2E can be used to determine bacteriostatic/bacteriolytic effect. It is not clear how such a distinction can be made from OD measurements, since an increase in OD can result from the entire population growing after removal of the stressor, or just part of the population that did not lyse/die. To make such a claim, the authors can spot bacteria on repressing media at different timepoints after protein induction, and then determine CFU.
    • Fig. 3A: A control is missing. To verify that the N-terminal part of Tde1 is not promiscuously interacting with proteins, the authors should include a control sample testing its inability to precipitate a protein other than Tap1 in the same experiment.
    • Fig. 3B: the blots are very "dirty". It is not clear how the authors were able to determine expression and precipitation of some truncations (for example, C2-Tde1 in the E. coli IP panel looks like a background band found in other lanes too).
    • Lines 222-225 (Fig. 4A): I can't see C-1-Tde1(M)-sfGFP in the cellular blot. All the bands in this lane look like background bands that are also present in all other lanes. Therefore, I am not sure how the conclusion regarding this truncation's ability to be secreted was reached.
    • Fig. 4A: the protein names above the lanes should include the sfGFP that is fused to them.
    • It would be preferable to show quantitative competition assays with statistics rather than pictures of a plate showing a single competition result, if conclusions or observations on minor differences in toxicity are made (for example, line 253: "The killing activity of Δtdei(Tde1GLGL-Tdi1) was largely compromised"). Since the authors performed each competition assay more than once, these data should be available to them.
    • Fig. 5A: The author claim at the beginning of the manuscript (first results section heading: "Tde1 can cause DNase-independent growth inhibition of prey cells") that the N-terminal region of Tde1 is toxic on its own in the prey cell, yet in this competition assay Tdi1(M) shows no toxicity against the E. coli target cells. In the microscopy assay (Fig. 4B), it appears that a lot of Tdi1(M) enters the prey cell, since we can visualize it under the microscope. Can the authors clarify this discrepancy and explain why they do not expect to see target killing by this mutant even though they claimed it is toxic earlier?
    • Fig. 5B: the GLGL mutant seems to have some residual toxicity, not dissimilar to what is shown in 5A. Why are these similar results interpreted differently (in 5A they are "largely compromised", while in 5B "killing activity... was not detectable")? Also, why was Tde(M)1-Tdi1 used in Fig. 5A but Tdi1(M) without the immunity gene used in Fig. 5B?
    • Fig. 5: Does the remaining third effector, Tae, not play a role in these competition assays? If, as shown in Fig. 5C, the entire T6SS is less active when a GLGL mutant is expressed, couldn't the different in toxicity shown in Figs. 5A-B be the result of lack of Tae secretion and toxicity?
    • Lines 359-362: T6SS effectors that bind the inner Hcp tube were suggested to be only partially folded.

    Significance

    The concept of T6SS effectors providing their own mechanism of transport from the cytoplasm to the periplasm is very interesting. It will appeal to audience in a wide range of microbiology disciplines, including those interested in toxins, membrane transport, and even translational applications. A similar concept was recently proposed and demonstrated for a domain that is also found in T6SS effectors (Atanaskovic et al., mBio, 2022).

    Expertise: I have been studying the different aspects of T6SS for the past decade.

  6. Version published to 10.1101/2022.07.12.499750v1 on bioRxiv