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The manuscript by Fridman et al. explores the unexpected finding that Aeromonas jandaei antagonistically employs a Type VI secretion system (T6SS) in a liquid environment. While researching the effector protein Awe1, which forms part of the T6SS apparatus, the authors observed T6SS-dependent intoxication of susceptible bacteria. Using a novel fluorescence-based screening method (named LiQuoR for liquid quantification of rivalry), the authors further determine that this intoxication is contact-dependent, and that contact between kin and non-kin Aeromonas bacteria in liquid is mediated by specific adhesins. Fridman et al. also identify additional marine bacteria capable of inflicting T6SS-mediated intoxication in liquid media, suggesting a mechanism for specific and contact-dependent bacterial competition and positing that such competition in liquid media may be more common in marine bacteria than previously documented. These findings have exciting implications for bacterial antagonism, potentially shifting the paradigm of how we view bacterial interactions in marine environments. We found this study to be well-written, containing high-quality data. Overall, the data presented in this manuscript are done well and support the claims made by the authors. We outline some major and minor adjustments aimed at aiding the clarity of reporting and presentation, strengthening the findings, as well as providing additional context for a broader audience.

Major Comments

- We are interested in the broader implications of the LiQuoR assay, particularly pertaining to this workflow’s application to different bacteria. The observation that the amount of prey luminescence in WT on solid media grew/increased after 4 h seemed counterintuitive to us (Figure 1E). It seems as if this result could make the workflow less sensitive for experiments done solely on solid media, further explanation of this finding would clarify on the workflows applicability to other solid surface experiments. Is this related to surface area? While this does not change the findings that inhibition is occurring in both liquid and in solid, it would enhance the clarity of these results to provide speculation on why this was seen.

- We are curious about your perspective on the observation that kin-kin aggregation facilitated by CaCl₂ supplementation does not increase kin intoxication but does increase non-kin intoxication (Figure 2A). Please speculate on this result in the discussion. Is the concentration used physiological?

- While the images shown in Figure 2B make it clear that aggregates are forming in liquid media, we have a suggestion to improve the strength of these results and account for the images not shown. For instance, quantification of the % of prey cells displaying Sytox staining would more strongly demonstrate the presence of permeabilized E. coli in multiple aggregates. This quantification could substitute Figure 2C (which can be moved into the supplemental): it was not totally clear to us why an orthogonal view was included here. If this is significant for the findings, it would increase clarity to include an explanation for an audience less familiar with this system.

-Lines 192-214: From a genomics perspective, we think further explaining how potential adhesins were identified would be helpful to increase the clarity and reproducibility of the experimental design. Please explain how you narrowed down these adhesins and located them in the genome, and why adhesins were targeted for this analysis over other proteins that could facilitate a physical interaction between predator and prey species. Define the acronyms and provide rationale for naming.

-Figure 6B nicely demonstrates that intoxication takes place in liquid between certain marine bacteria but not in Vpara. However, please include a control showing that V. para does intoxicate prey in solid media to strengthen these findings and confirm that this strain of V. para is capable of intoxicating prey under typical conditions.

-Given the significance of the TssB deletion for the core message of this work that type VI intoxication occurs in liquid media, please consider including data that confirm the TssB deletion e.g. sanger sequencing in supplemental or as source data. A complementation assay of TssB to show that regaining TssB restores the awe1 toxicity would be valuable.

- Lines 224-225/Figure 5: We are curious and excited about the implications of the balance between kin-aggregation and non-kin aggregation and how this may aid our understanding of bacterial interactions in marine environments. Based on our understanding of these results, the observation that deletion of CraAj (responsible for kin-kin aggregation) increased non-kin intoxication (mediated by LapAj) could suggest that aggregation between two kin cells, who both contain the needed immunity proteins, could dampen the intoxication of nearby non-kin cells. This result is implied by the data but not specifically speculated on or addressed. Though it may not be within the scope of this experimental design, our group was intrigued by these findings. Given your expertise in this area, consider discussing how these bacterial interactions may play out and/or include these observations as part of Figure 5.

Minor Comments

-All figures: In the legends, it is stated “these experiments were repeated three times with similar results”. Please define what is meant by an experiment e.g. technical or biological replicate.

-All figures: We felt that having the exact p-values indicating statistical significance is not necessary. For instance, in Figure 3B and 3D, we found it distracting that all of the values were significant by a factor of <1E-4, even when they appear different from each other. If this is simply a cutoff value, it would be helpful to keep that consistent between figures. Also, Figure 6A/B: The p-values presented, specifically the comparison between WT and T6SS – supplemented with 1 mM CaCl₂ (6A) and the two left hand panels of 6B, do not appear to match the differences shown between the experimental groups. By eye, these groups do not appear different from one another but are shown to be either highly statistically significant or not statistically significant at all.

- Figure 1A: To increase readability, we suggest that the colors could be more intuitive here- put WT in grey and then mix colors for double mutants. Bringing the light pink line (Δawei1 ΔtssB + pAwe1) to the front of the graph would further increase clarity.

-Figure 1B/F: Making color scheme consistent between 1B and 1F would increase clarity.

-LiQuoR assay: As there is often some level of variation in expression levels when working with a transformed population, confirmation that all prey strains luminesce to a similar level would provide further validation of this novel assay (similarly to what is done in FigS3B).

-Figure 2A: The colored box legends showing whether CaCl₂ is present or absent are inverted relative to one another, which we found to be confusing. To increase readability, please make them on the same side.

-Figure 3B,C,D,E: To help guide the eye on the graphs, we suggest adding dashed lines between each new mutation group (+/- TssB).

- Figure S1: Please include a loading control to verify assay input.

- Table S1: Clarify the gene and strain for each mutation.

- Line 112-113: It serves as an excellent control that the action of the T6SS apparatus is required for intoxication, however, since the T6SS apparatus is contained within the bacterium, would spent media contain free-floating T6SS proteins, or are these proteins only ejected from the bacterium in the presence of prey species? Please clarify. Direct evidence, such as immunoblotting, that effectors are present in the spent media from WT would make this claim more compelling.

- Line 35: While this part of the introduction provides excellent background regarding the role of T6SS in interactions with eukaryotic cells, it would be helpful to also specifically mention the role of T6SS in prokaryotic communities, as much of the later work focuses on competition between bacteria.

-Lines 70-71: A more thorough background on Aeromonas (lifestyle, importance, etc) is warranted.

-Line 84: Please provide the exact genotype when first introducing this mutant, it would improve clarity for the reader to explicitly state that this is a double mutant.

-Line 97: Clarify here that “Aj prey” in this paragraph refers to Aj which do not possess the cognate immunity protein, as the current phrasing could be interpreted to mean “prey of Aj”.

-Line 138: “Desired conditions for competition” is vague. Is solid media also incubated with shaking or is it static?

-Lines 156-157: The statement that all three effectors are injected into prey cells is broad and not necessarily supported within these findings. The injection of one effector could be favored, but other effectors could compensate in its absence.

-Line 189: Describes Aj as stably binding to other competing bacteria. To this point, imaged aggregates have been fixed so stability of aggregates may not be known.

-Line 248: Here, it is mentioned that there was a switch from using the Lux operon to using the RFP mCherry for improved cell detection. It might be helpful to clarify which fluorescent tag was used for each assay, as multiple different fluorescent tags are used.

-Line 317: As the choice to test CaCl₂ and the biological relevance of calcium for Aeromonas hosts is explained earlier in the manuscript, it would be interesting to include a brief explanation about the choice to include sodium chloride when assessing Vibrio intoxication rates. Presumably, sodium chloride was picked because Vibrio is commonly found in brackish water, but someone from outside the field may not be familiar with this biology. Additionally, since Aeromonas can be found in both fresh and brackish water, an interesting follow-up experiment would be to test the Aeromonasstrains under different salinities.

-Line 375-377: Needs citation.

-Line 385: Clarify “under specific conditions not addressed within the scope of this study”.

Carter Collins and Lily Pumphrey (Indiana University Bloomington) - not prompted by a journal; this review was written within a Peer Review in Life Sciences graduate course led by Alizée Malnoë with input from group discussion including Camy Guenther, Josy Joseph and Tahreem Zaheer. We are part of the Dept. of Biology where Julia Van Kessel’s group is located, Julia is a collaborator of the corresponding author and did not influence the choice of this preprint for our class.

Competing interests

The authors declare that they have no competing interests.

Use of Artificial Intelligence (AI)

The authors declare that they did not use generative AI to come up with new ideas for their review.