The type VI secretion system governs strain maintenance in a wild mammalian gut microbiome

Reviewer #1 (Public review):

Summary:

In this study, the authors investigate the physiological role of the Type VI secretion system (T6SS) in a naturally evolved gut microbiome derived from wild mice (the WildR microbiome). Focusing on Bacteroides acidifaciens, the authors use newly developed genetic tools and strain-replacement strategies to test how T6SS-mediated antagonism influences colonization, persistence, and fitness within a complex gut community. They further show that the T6SS resides on an integrative and conjugative element (ICE), is distributed among select community members, and can be horizontally transferred, with context-dependent effects on colonization and persistence. The authors conclude that the T6SS stabilizes strain presence in the gut microbiome while imposing ecological and physiological constraints that shape its value across contexts.

This study is likely to have a significant impact on the microbiome field by moving experimental tests of T6SS function out of simplified systems and into a naturally co-evolved gut community. The WildR system, together with the strain replacement strategy, ICE-seq approach, and genetic toolkit, represents a powerful and reusable platform for future mechanistic studies of microbial antagonism and mobile genetic elements in vivo.

The datasets, including isolate genomes, metagenomes, and ICE distribution maps, will be a valuable community resource, particularly for researchers interested in strain-resolved dynamics, horizontal gene transfer, and ecological context dependence. Even where mechanistic resolution is incomplete, the work provides a strong experimental foundation upon which such questions can be directly addressed.

Overall, this study occupies a space between system building and mechanistic dissection. The authors demonstrate that the T6SS influences persistence and community structure in vivo, but the physiological basis of these effects remains unresolved. Interpreting the results as evidence of fitness costs or selective advantage, therefore, requires caution, as multiple ecological and host-mediated processes could produce similar abundance trajectories.

Placing the findings within the broader literature on microbial antagonism, particularly work emphasizing measurable costs, benefits, and tradeoffs, would help readers better contextualize what is directly demonstrated here versus what remains an open question. Viewed in this light, the principal contribution of the study is to show that such questions can now be addressed experimentally in a realistic gut ecosystem.

Strengths:

A major strength of this study is that it directly interrogates the physiological role of the T6SS in a naturally evolved gut microbiome, rather than relying on simplified pairwise or in vitro systems. By working within the WildR community, the authors advance beyond descriptive surveys of T6SS prevalence and address function in an ecologically relevant context.

The authors provide clear genetic evidence that Bacteroides acidifaciens uses a T6SS to antagonize co-resident Bacteroidales, and that loss of T6SS function specifically compromises long-term persistence without affecting initial colonization. This temporal separation is well designed and supports the conclusion that the T6SS contributes to maintenance rather than establishment within the community.

Another strength is the identification of the T6SS on an integrative and conjugative element (ICE) and the demonstration that this element is distributed among, and exchanged between, community members. The use of ICE-seq to track distribution and transfer provides strong support for horizontal mobility and adds mechanistic depth to the study.

Finally, the transfer of the T6SS-ICE into Phocaeicola vulgatus and the observation of context-dependent colonization benefits followed by decline is a compelling result that moves the study beyond simple "T6SS is beneficial" narratives and highlights ecological contingency.

Weaknesses:

Despite these strengths, there is a mismatch between the precision of the claims and the precision of the measurements, particularly regarding fitness costs, physiological burden, and the mechanistic role of the T6SS.

First, while the authors conclude that the T6SS "stabilizes strain presence" and that its value is constrained by fitness costs, these costs are not directly measured. Persistence, abundance trajectories, and eventual loss are informative outcomes, but they do not uniquely identify fitness tradeoffs. Decline could arise from multiple non-exclusive mechanisms, including community restructuring, host-mediated effects, incompatibilities of the ICE in new hosts, or ecological retaliation, none of which are disentangled here.

Second, the manuscript frames the T6SS as having a defined physiological role, yet the data do not resolve which physiological processes are under selection. The experiments demonstrate that T6SS activity affects persistence, but they do not distinguish whether this occurs via direct killing, resource release, niche modification, or higher-order community effects. As a result, "physiological role" remains underspecified and risks being conflated with ecological outcome.

Third, although the authors emphasize context dependence, the study offers limited quantitative insight into what aspects of context matter. Differences between native and recipient hosts, or between early and late colonization phases, are described but not mechanistically interrogated, making it difficult to generalize beyond the specific cases examined.

Fourth is the lack of engagement with recent experimental literature demonstrating functional roles of the T6SS beyond simple interference competition. While the authors focus on persistence and competitive outcomes, they do not adequately situate their findings within recent work demonstrating that T6SS-mediated antagonism can serve additional physiological functions, including resource acquisition and DNA uptake, thereby linking killing to measurable benefits and tradeoffs. The absence of this literature makes it difficult to place the authors' conclusions about physiological role and fitness cost within the current conceptual framework of the field. Without this context, the physiological interpretation of the results remains incomplete, and alternative functional explanations for the observed dynamics are underexplored.

A further limitation concerns the taxonomic scope of the functional analysis. The authors state that the role of the T6SS in the murine environment is functionally investigated using genetically tractable Bacteroides species, citing the lack of genetic tools for Mucispirillum schaedleri. While this is a reasonable, practical choice, it means that a substantial fraction of T6SS-encoding species in the WildR community are not experimentally interrogated. Consequently, conclusions about the role of the T6SS in the murine gut necessarily reflect the subset of taxa that are genetically accessible and may not fully capture community-level or niche-specific functions of T6SS activity. Given that M. schaedleri is represented as a metagenome-assembled genome, its isolation and genetic manipulation would be technically challenging. Nonetheless, explicitly acknowledging this limitation and slightly tempering claims of generality would strengthen the manuscript.

Finally, several interpretations would benefit from more cautious language. In particular, claims invoking fitness costs, selective advantage, or physiological burden should be explicitly framed as inferences from persistence dynamics, rather than as direct measurements, unless supported by additional quantitative fitness or growth assays.

Reviewer #2 (Public review):

Summary:

In this study, the authors set out to determine how a contact-dependent bacterial antagonistic system contributes to the ability of specific bacterial strains to persist within a complex, native gut community derived from wild animals. Rather than focusing on simplified or artificial models, the authors aimed to examine this system in a biologically realistic setting that captures the ecological complexity of the gut environment. To achieve this, they combined controlled laboratory experiments with animal colonization studies and sequencing-based tracking approaches that allow individual strains and mobile genetic elements to be followed over time.

Strengths:

A major strength of the work is the integration of multiple complementary approaches to address the same biological question. The use of defined but complex communities, together with in vivo experiments, provides a strong ecological context for interpreting the results. The data consistently show that the antagonistic system is not required for initial establishment but plays a critical role in long-term strain persistence. This insight that moves beyond traditional invasion-based views of microbial competition. The observation that transferable genetic elements can confer only temporary advantages, and may impose longer-term costs depending on community context, adds important nuance to current understanding of microbial fitness.

Weaknesses:

Overall, there is not a lack of evidence, but a deliberate trade-off between ecological realism and mechanistic resolution, which leaves some causal pathways open to interpretation.

Reviewer #3 (Public review):

Summary:

Shen et al. investigate the contribution of the type VI secretion system of Bacteroidales in the gut microbiome assembly and targeting of closely related species. They demonstrate that B. acidifaciens relies on T6SS-mediated antagonism to prevent displacement by co-resident Bacteroidales and other members of the microbiome, allowing B. acidifaciens to persist in the gut.

Strengths:

Using a gnotobiotic model colonized with a wild-mouse microbiome is a significant strength of this study. This approach allows tracking of microbiome changes over time and directly examining targeting by Bacteroidales carrying T6SS in a more natural setting. The development of ICE-seq for mapping the distribution of the T6SS in the microbiome is remarkable, enabling the study of how this bacterial weapon is transferred between microbiome members without requiring long-read metagenomics methods.

Weaknesses:

Some conclusions are based on only four mice per condition. The author should consider increasing the sample size.

Overall, the authors successfully achieved their objectives, and their experimental design and results support their findings. As mentioned in the discussion, it would be important to investigate the role of the T6SS in resilience to disturbances in the microbiome, such as antibiotics, diet, or pathogen invasion. This work represents a step forward in understanding how contact-dependent competition influences the gut microbiome in relevant ecological contexts.

Author response:

We appreciate that the reviewers provided an overall positive assessment of our manuscript and offered constructive suggestions for improvement. All three reviewers noted that a key strength of our study is the implementation of a gut microbiome model for the characterization of interbacterial antagonism pathways such as the type VI secretion system (T6SS) that approaches natural complexity. They note our work represents a significant advance in microbiome research, and generates resources that will be of use to many researchers in the field. Two of the reviewers point out that the complexity of our model limits the nature of measurements we can make, and suggest we temper the strength of the some of the conclusions we draw. As noted in more detail below, in our revised manuscript, we will be more precise in the wording we use to characterize our findings, and we will be more explicit about what the measurements we are able to make allow us to conclude about the physiological role of the T6SS in the gut microbiome.

Reviewer #1 (Public review):

Summary:

In this study, the authors investigate the physiological role of the Type VI secretion system (T6SS) in a naturally evolved gut microbiome derived from wild mice (the WildR microbiome). Focusing on Bacteroides acidifaciens, the authors use newly developed genetic tools and strain-replacement strategies to test how T6SS-mediated antagonism influences colonization, persistence, and fitness within a complex gut community. They further show that the T6SS resides on an integrative and conjugative element (ICE), is distributed among select community members, and can be horizontally transferred, with context-dependent effects on colonization and persistence. The authors conclude that the T6SS stabilizes strain presence in the gut microbiome while imposing ecological and physiological constraints that shape its value across contexts.

This study is likely to have a significant impact on the microbiome field by moving experimental tests of T6SS function out of simplified systems and into a naturally co-evolved gut community. The WildR system, together with the strain replacement strategy, ICE-seq approach, and genetic toolkit, represents a powerful and reusable platform for future mechanistic studies of microbial antagonism and mobile genetic elements in vivo.

The datasets, including isolate genomes, metagenomes, and ICE distribution maps, will be a valuable community resource, particularly for researchers interested in strain-resolved dynamics, horizontal gene transfer, and ecological context dependence. Even where mechanistic resolution is incomplete, the work provides a strong experimental foundation upon which such questions can be directly addressed.

Overall, this study occupies a space between system building and mechanistic dissection. The authors demonstrate that the T6SS influences persistence and community structure in vivo, but the physiological basis of these effects remains unresolved. Interpreting the results as evidence of fitness costs or selective advantage, therefore, requires caution, as multiple ecological and host-mediated processes could produce similar abundance trajectories.

Placing the findings within the broader literature on microbial antagonism, particularly work emphasizing measurable costs, benefits, and tradeoffs, would help readers better contextualize what is directly demonstrated here versus what remains an open question. Viewed in this light, the principal contribution of the study is to show that such questions can now be addressed experimentally in a realistic gut ecosystem.

We thank the reviewer for this thoughtful summary of our study. We were glad to read they conclude our work will have a significant impact on the microbiome field and that the resources we have developed will be of value to the community.

Strengths:

A major strength of this study is that it directly interrogates the physiological role of the T6SS in a naturally evolved gut microbiome, rather than relying on simplified pairwise or in vitro systems. By working within the WildR community, the authors advance beyond descriptive surveys of T6SS prevalence and address function in an ecologically relevant context.

The authors provide clear genetic evidence that Bacteroides acidifaciens uses a T6SS to antagonize co-resident Bacteroidales, and that loss of T6SS function specifically compromises long-term persistence without affecting initial colonization. This temporal separation is well designed and supports the conclusion that the T6SS contributes to maintenance rather than establishment within the community.

Another strength is the identification of the T6SS on an integrative and conjugative element (ICE) and the demonstration that this element is distributed among, and exchanged between, community members. The use of ICE-seq to track distribution and transfer provides strong support for horizontal mobility and adds mechanistic depth to the study.

Finally, the transfer of the T6SS-ICE into Phocaeicola vulgatus and the observation of context-dependent colonization benefits followed by decline is a compelling result that moves the study beyond simple "T6SS is beneficial" narratives and highlights ecological contingency.

We appreciate this detailed and nuanced characterization of the strengths of our study.

Weaknesses:

Despite these strengths, there is a mismatch between the precision of the claims and the precision of the measurements, particularly regarding fitness costs, physiological burden, and the mechanistic role of the T6SS.

We acknowledge that in some places, our manuscript could benefit from greater precision in the language we use when linking the outcomes we observe in our study to their potential underlying causes. Specific revisions we propose to address this concern are described below.

First, while the authors conclude that the T6SS "stabilizes strain presence" and that its value is constrained by fitness costs, these costs are not directly measured. Persistence, abundance trajectories, and eventual loss are informative outcomes, but they do not uniquely identify fitness tradeoffs. Decline could arise from multiple non-exclusive mechanisms, including community restructuring, host-mediated effects, incompatibilities of the ICE in new hosts, or ecological retaliation, none of which are disentangled here.

We agree that multiple mechanisms could explain why P. vulgatus carrying a T6SS-encoding ICE declines over time. Our use of the term “fitness cost” to describe this trend was not meant to imply any particular underlying mechanism, but was rather our attempt to characterize the phenotypic outcome we observed in simplified terms. We note that ecological context is an important determinant of the fitness cost or benefit of any given trait, and our study sheds light on the importance of the presence of the WildR community and the mouse intestinal environment to the fitness contribution of the ICE to P. vulgatus. Nonetheless, to avoid implying an overly simplistic interpretation of our results, we propose to modify the language used in the manuscript when describing the contribution of the T6SS to species persistence in WildR-colonized mice.

Second, the manuscript frames the T6SS as having a defined physiological role, yet the data do not resolve which physiological processes are under selection. The experiments demonstrate that T6SS activity affects persistence, but they do not distinguish whether this occurs via direct killing, resource release, niche modification, or higher-order community effects. As a result, "physiological role" remains underspecified and risks being conflated with ecological outcome.

We acknowledge that our study does not fully resolve the physiological processes under selection that mediate role of the T6SS in maintaining B. acidifaciens populations in WildR-colonized mice. Indeed, several of the outcomes of T6SS activity the reviewer lists, such as target cell killing and nutrient release, are inextricably linked and thus inherently difficult to disentangle. We note that we did attempt to measure higher-order community effects of T6SS activity with metagenomic sequencing, but acknowledge that this approach may not have been sufficiently sensitive to detect small community shifts mediated by a relatively low-abundance species. To address the concern that our current framing implies more of a mechanistic understanding that our study achieves, we propose to substitute “ecological” for “physiological” where appropriate when summarizing our key findings.

Third, although the authors emphasize context dependence, the study offers limited quantitative insight into what aspects of context matter. Differences between native and recipient hosts, or between early and late colonization phases, are described but not mechanistically interrogated, making it difficult to generalize beyond the specific cases examined.

We are not entirely clear what the reviewer means by “differences between native and recipient hosts”, but we agree that additional quantitative studies will be needed to address the generalizability of our findings. Future studies are also needed to address the mechanistic basis for the difference in the benefit conferred by the T6SS that we observed between P. vulgatus and B. acidifaciens.

Fourth is the lack of engagement with recent experimental literature demonstrating functional roles of the T6SS beyond simple interference competition. While the authors focus on persistence and competitive outcomes, they do not adequately situate their findings within recent work demonstrating that T6SS-mediated antagonism can serve additional physiological functions, including resource acquisition and DNA uptake, thereby linking killing to measurable benefits and tradeoffs. The absence of this literature makes it difficult to place the authors' conclusions about physiological role and fitness cost within the current conceptual framework of the field. Without this context, the physiological interpretation of the results remains incomplete, and alternative functional explanations for the observed dynamics are underexplored.

We thank the reviewer for specifically highlighting the potential pertinence of this literature to our study. Indeed, we did not cite studies indicating a link between T6SS activity and the uptake of DNA and other resources released by targeted cells. As we note above, the release of intracellular contents from target cells is an inevitable consequence of the delivery of lytic effectors. Thus, distinguishing between fitness benefits conferred from the elimination of competitor species and those arising from scavenging the nutrients released during this process is not straightforward. Measuring the benefits deriving from the uptake of certain released molecules, such as DNA, was not immediately feasible in the system employed in this study and instead we focused on the direct lytic consequences of the effectors delivered via the T6SS. We will revise our Discussion to include reference to how downstream consequences of T6SS activity on target cells could impact the community, and thus the adaptive role of the T6SS in the microbiome.

A further limitation concerns the taxonomic scope of the functional analysis. The authors state that the role of the T6SS in the murine environment is functionally investigated using genetically tractable Bacteroides species, citing the lack of genetic tools for Mucispirillum schaedleri. While this is a reasonable, practical choice, it means that a substantial fraction of T6SS-encoding species in the WildR community are not experimentally interrogated. Consequently, conclusions about the role of the T6SS in the murine gut necessarily reflect the subset of taxa that are genetically accessible and may not fully capture community-level or niche-specific functions of T6SS activity. Given that M. schaedleri is represented as a metagenome-assembled genome, its isolation and genetic manipulation would be technically challenging. Nonetheless, explicitly acknowledging this limitation and slightly tempering claims of generality would strengthen the manuscript.

The reviewer points out that studying the T6SS activity in M. schadleri would potentially expand the generality of our claims. We agree that having an isolate of this species along with genetic tools for its manipulation would allow us to probe the importance of the T6SS in the gut microbiome more broadly. At the suggestion of the reviewer, we will add explicit mention for the need to develop such tools, an endeavor that lies outside of the scope of the current study.

Finally, several interpretations would benefit from more cautious language. In particular, claims invoking fitness costs, selective advantage, or physiological burden should be explicitly framed as inferences from persistence dynamics, rather than as direct measurements, unless supported by additional quantitative fitness or growth assays.

We agree with the reviewer that invoking fitness costs, selective advantages or physiological burdens should be done cautiously, and in our revised manuscript we will carefully re-evalute our usage of those terms. However, we would also argue invoking fitness costs and benefits when describe strain persistence dynamics in mice has substantial precedent in the literature ((Feng et al. 2020, Brown et al. 2021, Park et al. 2022, Segura Munoz et al. 2022), to list a handful of representative examples published by different groups). It is unclear to us what additional in vivo growth measurements could be taken to substantiate our claim that the T6SS provides a fitness benefit to B. acidifaciens during prolonged gut colonization, or that carrying the ICE imposes a fitness cost on P. vulgatus during long-term colonization. Our in vitro experiments evaluating the competitiveness conferred by T6SS activity provide a measure of insight into its fitness benefits, but as our in vivo strain persistence data and the work of many others show, in vitro measurements do not necessarily capture in vivo parameters.

Reviewer #2 (Public review):

Summary:

In this study, the authors set out to determine how a contact-dependent bacterial antagonistic system contributes to the ability of specific bacterial strains to persist within a complex, native gut community derived from wild animals. Rather than focusing on simplified or artificial models, the authors aimed to examine this system in a biologically realistic setting that captures the ecological complexity of the gut environment. To achieve this, they combined controlled laboratory experiments with animal colonization studies and sequencing-based tracking approaches that allow individual strains and mobile genetic elements to be followed over time.

Strengths:

A major strength of the work is the integration of multiple complementary approaches to address the same biological question. The use of defined but complex communities, together with in vivo experiments, provides a strong ecological context for interpreting the results. The data consistently show that the antagonistic system is not required for initial establishment but plays a critical role in long-term strain persistence. This insight that moves beyond traditional invasion-based views of microbial competition. The observation that transferable genetic elements can confer only temporary advantages, and may impose longer-term costs depending on community context, adds important nuance to current understanding of microbial fitness.

We thank the reviewer for the positive feedback and are glad they agree our study provides new insight into the role of interbacterial antagonism in natural communities.

Weaknesses:

Overall, there is not a lack of evidence, but a deliberate trade-off between ecological realism and mechanistic resolution, which leaves some causal pathways open to interpretation.

The reviewer makes a good point that the complexity of the experimental system we employ precludes some lines of experimentation that would yield more mechanistic information. As the reviewer notes, we were aware of the tradeoff between mechanistic resolution and ecological realism when selecting our experimental system. Our deliberate choice to favor biological complexity over mechanistic clarity in this study stemmed from our perception that a major gap in understanding of the T6SS and other antagonism pathways lies in defining their ecological function in complex microbial communities.

Reviewer #3 (Public review):

Summary:

Shen et al. investigate the contribution of the type VI secretion system of Bacteroidales in the gut microbiome assembly and targeting of closely related species. They demonstrate that B. acidifaciens relies on T6SS-mediated antagonism to prevent displacement by co-resident Bacteroidales and other members of the microbiome, allowing B. acidifaciens to persist in the gut.

Strengths:

Using a gnotobiotic model colonized with a wild-mouse microbiome is a significant strength of this study. This approach allows tracking of microbiome changes over time and directly examining targeting by Bacteroidales carrying T6SS in a more natural setting. The development of ICE-seq for mapping the distribution of the T6SS in the microbiome is remarkable, enabling the study of how this bacterial weapon is transferred between microbiome members without requiring long-read metagenomics methods.

We thank the reviewer for their enthusiasm toward our study.

Weaknesses:

Some conclusions are based on only four mice per condition. The author should consider increasing the sample size.

We agree that in some experiments it would be beneficial to increase the sample size from four mice. However, the experiments we performed for this study are time and resource intensive. Additionally, the experiments on which we base our primary conclusions were all independently replicated with similar results. Given these factors, we determined that the extra confidence that might be afforded by increasing our sample size did not merit the delay in publication and investment in resources that would be required.

Overall, the authors successfully achieved their objectives, and their experimental design and results support their findings. As mentioned in the discussion, it would be important to investigate the role of the T6SS in resilience to disturbances in the microbiome, such as antibiotics, diet, or pathogen invasion. This work represents a step forward in understanding how contact-dependent competition influences the gut microbiome in relevant ecological contexts.

We agree that investigating the role of the T6SS during perturbations of the microbiome is a key next step for this work and thank the reviewer for highlighting this important future direction.

References

Brown, E. M., H. Arellano-Santoyo, E. R. Temple, Z. A. Costliow, M. Pichaud, A. B. Hall, K. Liu, M. A. Durney, X. Gu, D. R. Plichta, C. A. Clish, J. A. Porter, H. Vlamakis and R. J. Xavier (2021). "Gut microbiome ADP-ribosyltransferases are widespread phage-encoded fitness factors." Cell Host Microbe 29(9): 1351-1365 e1311.

Feng, L., A. S. Raman, M. C. Hibberd, J. Cheng, N. W. Griffin, Y. Peng, S. A. Leyn, D. A. Rodionov, A. L. Osterman and J. I. Gordon (2020). "Identifying determinants of bacterial fitness in a model of human gut microbial succession." Proc Natl Acad Sci U S A 117(5): 2622-2633.

Park, S. Y., C. Rao, K. Z. Coyte, G. A. Kuziel, Y. Zhang, W. Huang, E. A. Franzosa, J. K. Weng, C. Huttenhower and S. Rakoff-Nahoum (2022). "Strain-level fitness in the gut microbiome is an emergent property of glycans and a single metabolite." Cell 185(3): 513-529 e521.

Segura Munoz, R. R., S. Mantz, I. Martinez, F. Li, R. J. Schmaltz, N. A. Pudlo, K. Urs, E. C. Martens, J. Walter and A. E. Ramer-Tait (2022). "Experimental evaluation of ecological principles to understand and modulate the outcome of bacterial strain competition in gut microbiomes." ISME J 16(6): 1594-1604.