Agrobacteria deploy two classes of His-Me finger superfamily nuclease effectors exerting different antibacterial capacities against specific bacterial competitors

  1. Mary Nia M. Santos
  2. Katherine L. Pintor
  3. Pei-Yu Hsieh
  4. Yee-Wai Cheung
  5. Li-Kang Sung
  6. Yu-Ling Shih
  7. Erh-Min Lai

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Abstract

The type VI secretion system (T6SS) assembles into a contractile nanomachine to inject effectors across bacterial membranes for secretion. The Agrobacterium tumefaciens species complex is a group of soil inhabitants and phytopathogens that deploys T6SS as an antibacterial weapon against bacterial competitors at both inter-species and intra-species levels. The A. tumefaciens strain 1D1609 genome encodes one main T6SS gene cluster and four vrgG genes (i.e., vgrGa-d ), each encoding a spike protein as an effector carrier. A previous study reported that vgrGa- associated gene 2, named v2a, encodes a His-Me finger nuclease toxin (also named HNH/ENDO VII nuclease), contributing to DNase-mediated antibacterial activity. However, the functions and roles of other putative effectors remain unknown. In this study, we identified vgrGc- associated gene 2 ( v2c ) that encodes another His-Me finger nuclease but with a distinct Serine Histidine Histidine (SHH) motif that differs from the AHH motif of V2a. We demonstrated that the ectopic expression of V2c caused growth inhibition, plasmid DNA degradation, and cell elongation in Escherichia coli using DNAse activity assay and fluorescence microscopy. The cognate immunity protein, V3c, neutralizes the DNase activity and rescues the phenotypes of growth inhibition and cell elongation. Ectopic expression of V2c DNase-inactive variants retains the cell elongation phenotype, while V2a induces cell elongation in a DNase-mediated manner. We also showed that the amino acids of conserved SHH and HNH motifs are responsible for the V2c DNase activity in vivo and in vitro . Notably, V2c also mediated the DNA degradation and cell elongation of the target cell in the context of interbacterial competition. Importantly, V2a and V2c exhibit different capacities against different bacterial species and function synergistically to exert stronger antibacterial activity against the soft rot phytopathogen, Dickeya dadantii .

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  1. Version published to 10.3389/fmicb.2024.1351590
  6. Version published to 10.1101/2023.03.20.533438v2 on bioRxiv
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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    The article describes a study on two effector nucleases, V2c and V2a, encoded by the T6SS cluster in Agrobacterium tumefaciens 1D1609. The study shows that V2c is a DNase belonging to the Tox-SHH clade of His-Me finger superfamily, exhibiting DNase activity in vivo and in vitro. The SHH and HNH motif of V2c were found to be involved in DNase activity. The study also demonstrates that V2c induces DNA degradation and cell elongation, which can be neutralized by its cognate immunity protein V3c. Furthermore, V2a, also exhibits DNase activity-dependent cell elongation phenotype. Both V2a and V2c nucleases function synergistically for antibacterial activity against Dickeya dadantii, resulting in elongated and lysed cells. The study suggests that 1D1609 uses V2a and V2c DNase effectors with synergistic antibacterial activity against Dickeya dadantii.

    Major issues:

    1. The cell elongation phenotype was an important focus of the paper and the authors did a solid job quantifying this phenotype. However, cell elongation does not seem to be associated with the mechanism of toxicity. Just a small part of the cells are elongated while you have more than one log of killing (Figure 5C and 5D).
      Many stresses can result in cell elongation, such as cell-wall targeting antibiotics. The signal narrows down to the very well-known mechanism of SulA activated by SOS response. There is no evidence "cell elongation independent of nuclease activity may represent a new mechanism of stress response #339". I strongly suggest the authors to use an SOS like pPrecA-gfp from ref 17 if they want to invest in the cell elongation phenotype. As it is, cell elongation is a distraction from the most exciting things in the manuscript. One potential hypothesis is that the catalytic mutants may bind DNA without cleaving, and while bound to the DNA, the mutants may interfere with DNA metabolism, replications, transcription, etc... This interference could create breaks in the DNA and cause cell wall elongation.
    2. The manuscript does not have biological replicates in many experiments. At first glance, the tiny error bars in many graphs raised a red flag. It is very difficult, if not impossible, to have the date so tight when working with bacterial cultures. Many of these graphs have "independent experiments", but in Fig 5 the authors mention " 6 repeats from three independent experiments". My understanding is that the independent experiments are from the same cultures, which makes them technical and not biological replicates. The authors need to have replicates from independent cultures. I ask the authors to explain better what they mean by replicates and their rationale.

    Minor issues:

    1. Many figures have all the data with conditions with and without arabinose. This makes the figure very polluted and difficult to follow. I suggest the authors keep only the data from induced cultures and move the figures with all the data to supplement. This is a big issue with Fig 3
    2. The entire "V2c V2a nucleases function synergistically for antibacterial activity against the soft rot phytopathogen, Dickeya dadantii" #212 section is difficult to follow because the name of the toxins are not in the name of the strains. The reader should be able to associate the toxin with the strain.
    3. The in vitro essay #427 should have information about the amount of enzyme used.
    4. The competition essay #474 method does not have enough information to allow reproducibility and must be expanded.
    5. Figure 2B Y-axis needs one log increase between marks, and not two.
    6. #256 is difficult to understand and not very scientific. DNses are potent because they target essential molecules, the same for lipases and muramidases. This is not related to the origin of life.

    Significance

    Strengths:

    Data is clear about the toxicity and DNA degradation phenotypes The synergy of toxins is a very exciting topic; it helps to explain why some strains have redundant toxins

    Weakness:

    Cell elongation claims are not supported Replicates are inadequate Methods needs to be more specific

    The manuscript provides incremental data about bacteria-to-bacteria toxins. The major finding of predicted nuclease acting as nuclease toxins is not particularly innovative. This work will benefit a smaller audience in the field of toxins.

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    Referee #2

    Evidence, reproducibility and clarity

    Santos et al demonstrate the activity and confirm predictions of the mechanism of action of SHH nuclease toxin encoded in auxiliary T6SS cluster of Agrobacterium tumefaciens. Study includes demonstration of nuclease activity in vitro and its manifestation in vivo, mutational analysis of the predicted catalytic site, and assessment of its role in inter-bacterial competition using strains deleted for the T6SS effector(s).

    Major comments: major issues affecting the conclusions

    Lines 159 and further - I am not sure that large deletion as an entire region between residues 380-409 is a very healthy approach. Without showing that such protein can be produced and fold, it can be very risky to draw any conclusions. While expression of other mutants are shown in Figure 2D, this construct is not included. Also, it is not clear (starting from line 160) what is the utility of double catalytic mutant if single mutants are already inactive. Was there residual activity of single mutants after all?

    Line 187 and Figure 3 - cell elongation of CR mutant and HAHA mutant seems to be independent of expression of the construct. It is therefore incorrect to write that "E. coli expressing ...", since theoretically it should not express anything in the absence of arabinose. Also, how do would authors interpret these findings? Again, at Line 211 - in the same paragraph authors say "V2c shows DNase activity-independent cell elongation" and then conclude "cell elongation phenotype may be specific to toxicity of nuclease effectors". These two phrases seem to contradict each other.

    Line 272 on - authors speculate about dependency on the metals, which is a hallmark of the his-me finger nucleases. A simple test could have been adding the EDTA control to chelate the metal in the in vitro experiment such as one presented in figure 2D. (OPTIONAL)

    Minor comments

    Figure 1 - Auxiliary cluster with accession numbers, in addition to the domain composition of the toxin could be demonstrated. This would help the readers to identify which effector is studied and link it to other studies.

    Line 127 and Figure 1 C and line 303 - the last option, named "FIX RhsA AHH HNH-like" seems to be a mix of multiple things, First, FIX domain is already included as a first option; AHH HNH-like corresponds to toxic domain (although it often ends up in annotation of the entire protein). All His-Me finger nucleases at some point were annotated as HNH-like and thus HNH, AHH, SHH, ... all belong to the same clade of nuclease domains; RhsA is probably a correct domain/protein detected here and to be represented here as a separate option. I would call it "Rhs", not RhsA, since RhsA,B,C,... is part of an historical systematics from E.coli, but is probably true for one strain only, since Rhs ends (C-terminal toxic domains) are highly variable between species and even strains. To my knowledge, Rhs do encode AHH-like domains and quite often. In conclusion, this is just a mess of protein naming that was picked up from databases, I would correct this name for "Rhs".

    Line 181-182 - text is speaking about v2c H383A v2c H384A, but in figure 3, it is v2c HAHA. It might be just naming differences, but it should be consistent.

    Line 246 - expression "detoxify D. dadantii" is unclear and a little confusing here, did authors mean kill or eliminate?

    Line 263 - "that consisting of" should be "that comprise" or "that possess"

    Referees cross-commenting

    I agree with the reviewer #1 that the text could be improved by better explaining the nomenclature, and reasoning behind certain experiments such as choice of prey cells. Regarding the novelty, to my opinion it is a choice of authors - either limit their study as it is now and it does not stand out by neither approach nor subject, or as suggested by the reviewers explore the mode of action, specificity and the reason behind the cell filamentation.

    I agree with the reviewer #3 that the cell elongation seems to be central interest but it is not investigated properly. I do think, that the SOS reporter would strengthen the study and would help to support (or not) some of the statements. I appreciate the scrutiny of reviewer #3 and I agree that the replicates seem to have extremely low variation and authors should provide precise explication on the reproducibility.

    Overall, I agree with the two other reviewers that the weakness of this manuscript is lack of innovation and to some extent lack of support for certain claims. The study could be improved by making it more profound, but a lot of additional work will be needed to bring it to another level.

    Significance

    Overall, this study is rather detailed, but not very novel - SHH and other HNH nucleases have been already assessed in literature using very similar methods, even by the same authors (Santos et al., Front Microbiol 2020). On the other hand, the study presents in depth investigation of auxiliary toxin, but shows that it is fully functional and has a role in killing, which is important and interesting for the field of Agrobacterium.

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    Referee #1

    Evidence, reproducibility and clarity

    This study characterised one of the four previously identified T6SS effectors of Agrobacterium tumefaciens strain 1D1609. The effector, named V2c, is a His-Me finger nuclease likewise the previously characterised V2a, although V2c has a distinct SHH motif. V2c was found to induce growth inhibition, plasmid degradation, and cell elongation in E. coli. The cognate immunity protein V3c can neutralise the DNase activity of V2c. V2a and V2c are found to function synergistically to exhibit stronger antibacterial activity against a phytopathogen, Dickeya dadantii.

    Although the manuscript provides a decent characterisation of the T6SS effector V2c of strain 1D1609 of A. tumefaciens, there are several areas where the authors could make significant improvements to their work.

    • The introduction section of the manuscript would benefit from a more detailed background of the research to situate the reader in the appropriate context for a better understanding of the results of this study. Specifically, the authors could provide more information regarding the four effectors of A. tumefaciens 1D1609, their domains, their genetic context, and their immunity proteins. In fact, three of the effectors are, to a different extent, named in the manuscript, whereas the fourth one is not mentioned at all. The authors should also aim to provide more context and explanation of the nomenclature of the effectors. This will make the paper more understandable for readers unfamiliar with the terminology of Agrobacterium effectors. In addition, the authors should consider giving more attention to the phenotype of previously described nucleases, such as cell elongation, so that the reader would better understand the result section when encountering this phenotype. The authors only explain this in the discussion, and it would be helpful to have more clarification in the Introduction as well. The authors should also explain the reasoning behind using that prey cell (Dickeya) and not E. coli or another organism for the antibacterial activity experiments.
    • The novelty of the study could be improved by providing a better explanation of the specific mechanism or mode of action through which V2c works. For example, the authors could study more deeply the puzzling fact that the elongation phenotype is independent of the nuclease activity for this effector but not for V2a. Another interesting approach would be to study the putative specificity of these nuclease effectors, as not all of them are effective against bacterial targets. This is an unexplored area of great interest for a more innovative study because nucleases do not seem to be specific, they all degrade nucleic acids, and somehow they have different capacities to kill different prey cells.
    • In Figure 1B, it would be convenient to include in the alignment the two nucleases of the same family as V2c that have been already described in the literature and are named in the main text (Tke4 and Txe4) to better illustrate their similarities and differences. In Figure 1C, it would be convenient to add the PAAR-RHS domain to the list of N-terminal domains found with Tox-SHH nucleases.
    • When it comes to the microscopy experiments (Figure 5), the authors should work to improve the relevance and quantification of the data they present. This could involve using more rigorous analysis techniques, such as statistical analysis, to support their findings more convincingly. In fact, the authors could enhance the rigour of their analysis (Dickeya cell lysis) by gathering more supporting evidence before drawing their conclusions. The authors should explain why they are using two identical strains (attackers) in the competition assays (Fig 5C) d3EIbcd 1 and 2.
    • Regarding the inter-bacterial competition settings between Agrobacterium and Dickeya, the authors should explain why they used TssB-GFP prey cells when this is not necessary for the assays they performed. Furthermore, the authors should clarify the statement in the discussion (lane 353-355) regarding the absence of antibacterial activity of V2c against E. coli, while the results of this work show inhibition of E. coli cells (Fig. 2). The authors should rethink whether the video is necessary for this section, as it does not provide additional relevant information.
    • To ensure that the paper is easily comprehensible and effectively conveys its message, the authors should meticulously examine all aspects of their writing, including language usage, grammatical accuracy, and syntax structure. The authors should also ensure consistency in their writing style and language usage throughout the paper. As one example of writing style inconsistency, the authors used both Gram-negative and gram-negative in the manuscript.

    Significance

    The study expands the knowledge in the specific field of SHH nucleases and T6SS effectors. This could be of interest to T6SS researchers and more broadly to researchers in the field of bacterial toxins.

    The novelty of the study is very limited since the authors functionally characterised a T6SS effector with a well-described function, a DNAse (DNA degradation) and a recognised structure (SHH domain). As expected for a nuclease, it degrades DNA and provokes cell elongation, which has also been described before.

  11. Version published to 10.1101/2023.03.20.533438v1 on bioRxiv