# Antagonism as a foraging strategy in microbial communities

Astrid Stubbusch, François Peaudecerf, Kang Soo Lee, Lucas Paoli, Julia Schwartzman, Roman Stocker, Marek Basler, Olga Schubert, Martin Ackermann, Cara Magnabosco, et al.

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## To cite this version:

Astrid Stubbusch, François Peaudecerf, Kang Soo Lee, Lucas Paoli, Julia Schwartzman, et al. *Antagonism as a foraging strategy in microbial communities*. Science, 2025, 388 (6752), pp.1214-1217.
10.1126/science.adr8286. hal-05120063

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Submitted on 19 Jun 2025

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<figure><img src="image_2.png" /></figure>
Antagonism as a foraging strategy in microbial communities

Astrid K.M. Stubbusch<fnref n="1" />,<fnref n="2" />,<fnref n="3" />*†, François J. Peaudecerf<fnref n="4" />,<fnref n="5" />, Kang Soo Lee<fnref n="6" />,<fnref n="5" />, Lucas Paoli<fnref n="7" />,<fnref n="8" />, Julia Schwartzman<fnref n="9" />, Roman Stocker<fnref n="5" />, Marek Basler<fnref n="10" />, Olga T. Schubert<fnref n="1" />,<fnref n="2" />, Martin Ackermann<fnref n="1" />,<fnref n="2" />,<fnref n="11" />‡, Cara Magnabosco<fnref n="3" />‡, Glen G. D'Souza<fnref n="1" />,<fnref n="2" />,<fnref n="12" />‡.

<footnote n="1">¹Institute of Biogeochemistry and Pollutant Dynamics, Department of Environmental Systems Science, ETH Zurich; Zurich, Switzerland.</footnote>

<footnote n="2">²Department of Environmental Microbiology, Eawag: Swiss Federal Institute of Aquatic Science and Technology; Dübendorf, Switzerland.</footnote>

<footnote n="3">³Geological Institute, Department of Earth and Planetary Sciences, ETH Zurich; Zurich, Switzerland.</footnote>

<footnote n="4">⁴Univ Rennes, CNRS, IPR (Institut de Physique de Rennes) - UMR 6251, Rennes, France.</footnote>

<footnote n="5">⁵Institute of Environmental Engineering, Department of Civil, Environmental and Geomatic Engineering, ETH Zurich; Zurich, Switzerland.</footnote>

<footnote n="6">⁶Department of Mechanical Engineering, Ulsan National Institute of Science and Technology (UNIST); Ulsan, South Korea. Ulsan, South Korea.</footnote>

<footnote n="7">⁷Department of Biology, Institute of Microbiology and Swiss Institute of Bioinformatics, ETH Zurich; Zurich, Switzerland.</footnote>

<footnote n="8">⁸Global Health Institute, School of Life Sciences, École Polytechnique Fédéral de Lausanne (EPFL); Lausanne, Switzerland</footnote>

<footnote n="9">⁹Department of Biological Sciences, University of Southern California; Los Angeles, USA.</footnote>

<footnote n="10">¹⁰Biozentrum, University of Basel; Basel, Switzerland.</footnote>

<footnote n="11">¹¹Laboratory of Microbial Systems Ecology, School of Architecture, Civil and Environmental Engineering (ENAC), École Polytechnique Fédéral de Lausanne (EPFL); Lausanne, Switzerland.</footnote>

<footnote n="12">¹²Center for Fundamental and Applied Microbiomics, Biodesign Institute, Arizona State University, Tempe, AZ, USA.</footnote>

<footnote n="13">¹³School of Molecular Sciences, Arizona State University, Tempe, AZ, USA.</footnote>

<footnote>*Corresponding author. Email: <astubbusch@icloud.com></footnote>

<footnote>†Present address: Department of Microbiology, Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia.</footnote>

<footnote>‡These authors contributed equally to this work.</footnote>
**Abstract**

In natural habitats, nutrient availability limits bacterial growth. We discovered that bacteria can overcome this limitation by acquiring nutrients through lysing neighboring cells via contact-dependent antagonism. Using single-cell live imaging and isotopic markers, we found that during starvation the type VI secretion system (T6SS) lysed neighboring cells, and thus provided nutrients from lysing cells for growth. Genomic adaptations in antagonists, characterized by a reduced metabolic gene repertoire, and the previously unexplored distribution of the T6SS across bacterial taxa in natural environments suggest that bacterial antagonism may contribute to nutrient transfer within microbial communities in many ecosystems.

**Main Text**

Bacteria require nutrients to synthesize cellular building blocks, such as nucleotides, amino acids and fatty acids, for growth. In nature, nutrients are often found only at low concentrations or in the form of complex polymers that require specific enzymatic degradation before they can be used as nutrients. Under such conditions, the biomass of other cells, rich in common metabolites and cellular building blocks, could serve as a valuable nutrient source (1). Killing neighboring cells can give access to these nutrients.

Bacteria have evolved various systems to lyse neighboring bacteria by transferring toxins, such as the type IV and type VI secretion systems (T4SS and T6SS) (2–4). These contact-dependent antagonistic systems are widespread in cultured bacteria (3). Their dominant ecological role is usually viewed as mediating competition for space and nutrients (5–7), leading to their characterization as ‘weapons’ of bacterial ‘warfare’ (7, 3, 8–11).

In this study, we ask whether contact-dependent antagonism mechanisms enable bacteria to acquire nutrients from target cells, in addition to their role in killing competitors. This has been hypothesized previously (12, 13), but a direct role in nutrient foraging has never been shown experimentally for non-predatory bacteria. We focus on the T6SS, a well-studied bacteriophage-like molecular machinery that enables transfer of diverse toxins into neighboring cells and that is present in approximately 25% of all sequenced Gram-negative bacteria (14) though the distribution of T6SS+ taxa across natural environments remains uncharted.

**Contact-dependent antagonism enables growth during starvation**

To determine whether the biomass of target cells provides a source of nutrients for antagonistic cells, we studied a simple community consisting of the marine isolates *Vibrio cyclitrophicus* ZF270 (target cells) and the T6SS-encoding *Vibrio anguillarum* FS144 (T6SS+ cells) as an ecological model system. We investigated the growth of T6SS+ cells and the lysis of target cells at single-cell resolution within a microfluidic device, where cells can grow in a
monolayer in shallow microfluidic chambers (fig. S1). When we provided the T6SS+ cells with a carbon source that they can metabolize (N-acetylglucosamine (GlcNAc), fig. S2), the cells grew exponentially with a doubling rate of ~0.2 h⁻¹ (Fig. 1A, 1B, fig. S3, Movie S1). The doubling rate is a measure that is independent of the initial number of cells per chamber, which varies among chambers (fig. S3). In coculture with target cells, we observed a similar doubling rate (Fig. 1B, fig. S3). When we provided a carbon source that only the target cells could metabolize (alginate, fig. S2), T6SS+ cells failed to multiply in monoculture but were able to grow in coculture with target cells at a doubling rate of ~0.07 h⁻¹ (Fig. 1B, fig. S3, Movie S2 and 3).

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** Growth benefit and acquisition of nutrients released through contact-dependent antagonism via the T6SS. (A) Representative images of T6SS+ cells (V. anguillarum FS144, cyan) and target cells (V. cyclitrophicus ZF270, magenta) supplied with different carbon sources in microfluidic chambers at the start (0 h) and end time point (12 h, 24 h, and 24 h, top to bottom, respectively, based on the time it took for V. anguillarum cells to fill the chamber) (Scale bar: 5 µm). (B) Growth rate of T6SS+ cells (V. anguillarum FS144) in microfluidic chambers estimated from the exponential increase of cell numbers over time shown in fig. S3. Points show the estimates from individual growth chambers (fig. S3) and box plots indicate the median and interquartile range (n = 6, 8, 5, 6; asymptotic Wilcoxon-Mann-Whitney tests, *p* = 0.04). (C) Isotope incorporation by V. cholerae without T6SS (light blue) and with T6SS (dark blue) from deuterium-labeled E. coli (yellow). The deuterium signal in individual V. cholerae cells was quantified using Raman microspectroscopy. The coculture medium contained deuterium and melibiose as carbon source for continued growth of labeled E. coli. The values were normalized by subtracting the signal of the respective V. cholerae cells in this medium in monoculture (fig. S5E). T6SS illustrated as red spikes, deuterium as star symbols (n = 40, 35; Wilcoxon rank-sum tests, *p* = 8 x 10⁻¹³).</figcaption></figure>

To investigate whether the growth of T6SS+ cells without a metabolizable carbon source is facilitated by the T6SS-mediated killing of target cells, rather than the exchange of secreted nutrients (cross-feeding), we tested whether the growth is contingent on a functional T6SS. We used a genetic model system, namely *V. cholerae* 2740-80 and its T6SS-
deletion mutant (15, 16), paired with *E. coli* as target cells. Again, we provided T6SS+ cells and target cells in microfluidic chambers with a carbon source that T6SS+ cells could not metabolize (melibiose, fig. S1, Movie S4). We found that the growth rate of the T6SS-deletion mutant population was lower than that of the T6SS+ population (fig. S4 and S5A and B, Movie S5 and S6), suggesting that T6SS-mediated cell lysis increased the growth of T6SS+ cells under carbon starvation conditions. This finding was confirmed by growth rate measurements in bulk culture experiments (-0.1 h⁻¹ and 0.3 h⁻¹ for the T6SS-deletion population and T6SS+ population, respectively; fig. S5C).

To test whether the observed growth increase in T6SS+ cells is caused by the acquisition of nutrients from target cells, we used stable isotope probing (SIP)-Raman microspectroscopy (17). We first labeled *E. coli* target cells with deuterium and then cocultured them with unlabeled *V. cholerae* T6SS+ cells or the T6SS-deletion mutants. After 9 hours, T6SS+ cells showed significant levels of deuterium incorporation compared to the T6SS-deletion cells (Fig. 1C, fig. S5D-E), further confirming that the T6SS+ is indeed facilitating access to nutrients.

## Slow cell lysis increased the nutrient uptake by surrounding cells

A more detailed examination of T6SS-driven antagonism revealed that lysis is not instantaneous. We observed that the cell shape of attacked target cells changed from rod-shaped to round before bursting, likely because T6SS effectors compromised cell wall integrity (18) (table S2 and S3). These round *V. cyclitrophicus* target cells persisted for 1.43 ± 0.20 h in alginate medium and 0.33 ± 0.09 h in GlcNAc medium before bursting (mean ± 95% confidence interval) (Fig. 2A). Staining cells with propidium iodide (PI), which does not permeate intact cell membranes but can enter cells with leaky membranes (19, 20), we found that round target cells incorporated more PI than target cells in their usual rod shape (Fig. 2B). Together, these findings suggest that the action of carbon-starved T6SS+ cells causes slow lysis of target cells, likely leading to gradual rather than instantaneous nutrient release.

To test whether slow cell lysis improves nutrient acquisition from target cells, we modeled the nutrient concentration field of a cell that bursts instantaneously in comparison with that of a cell that releases the same amount of nutrients slowly over time (Fig. 2C). We then modeled the nutrient uptake rate of a neighboring T6SS+ cell using Michaelis-Menten kinetics to take into account that membrane transport can reach saturation. Considering the typical intracellular concentration $C_i$ of amino acids and the half-saturation constant $K_d$ of amino acid transporters of *E. coli* (21, 22), we estimated that the approximate total nutrient gain of T6SS+ cells by slow lysis is 2-fold to 50-fold greater than that through fast lysis (Fig. 2C and D). Fast lysis led to larger initial concentrations of nutrients but exceeded the uptake capacity of the T6SS+ cell's transporters, thus limiting its uptake. T6SS+ cells would acquire more nutrients from a bursting target cell than a slowly lysing target cell only if the target
cell's $C_i$ was over 100-fold smaller or the $K_d$ was over 10-fold higher than for amino acids in E. coli. These results suggest that T6SS+ cells gain more nutrients from slow cell lysis.

<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Slow cell lysis increases the nutrient gain for T6SS cells. (A) *V. anguillarum* T6SS cells lyse the *V. cyclitrophicus* target cells slowly, especially under starvation conditions in alginate medium. (B) Induction of a round shape in target cells is associated with leakiness of the cell membrane, estimated by the propidium iodide (PI) staining intensity of round (length-to-width ratio of 1 to 1.8) and rod-shaped (length-to-width ratio of >1.8 to 6) target cells. Each dot represents the mean value from one microfluidic chamber (from 2 independent experiments with 6 chambers each). Jitter was applied in the horizontal direction (n = 12, 12; Wilcoxon signed-rank exact test, *p* = 5 x 10⁻⁴). (C) Schematic diagram of the mathematical model that compares the nutrient concentration field available to T6SS cells through fast lysis (top) or slow lysis (bottom), alongside the nutrient release from the target cell over time (right). (D) Model predictions comparing the approximated total nutrient uptake by an antagonistic cell through slow and fast cell lysis, suggesting slow lysis increases nutrient uptake in the physiological parameter range. For slow lysis, we assume that the surface concentration of leaked metabolites is small compared to $K_d$ of the T6SS cell. The green box depicts literature values for $C_i$ and $K_d$ in E. coli (table S4).</figcaption></figure>
We found that the conversion of biomass from slowly lysing cells into new T6SS+ cells is efficient. In densely populated microfluidic chambers, on average one new T6SS+ cell formed for every 2-3 lysing target cells (fig. S6B). This biomass conversion is substantially more efficient than previously reported for various taxa in well-mixed systems, which required 13 to 2,000 heat-killed cells for the growth of one new cell (23). This high efficiency may partly be explained by the approximately 2-fold larger volume of the target cells compared to the T6SS+ cells in our system (fig. S6C to E). However, it also suggests that T6SS+ cells more efficiently acquire nutrients from slowly lysing cells in sessile microbial communities.

## Environmental significance of the employment of the T6SS for nutrient foraging

If T6SS are generally used to generate sources of nutrients by lysing cells, one would expect to observe a reduction of genes needed for the degradation of complex organic matter and synthesis of cellular building blocks, as observed in pathogenic and predatory bacteria (24, 25). To test this idea, we compared the gene repertoire of T6SS positive and T6SS negative genomes within the genus Vibrio. We analyzed ~6,000 publicly available high-quality genomes. First, we screened the genomes for the presence of a T6SS (fig. S7) and then de-replicated the genomes into 141 operational taxonomic units (OTUs) (fig. S8).

We found T6SS+ genomes in 82 OTUs, genomes without T6SS in 39 OTUs, and 20 OTUs that possessed both T6SS+ and T6SS- genomes (fig. S9). A pan-genome-wide association study (26) on representative genomes of the 82 T6SS+ and 39 T6SS- OTUs was used to identify T6SS associated genes. As many bacterial traits are conserved among related taxa, we removed associations that arose from the underlying phylogenetic structure of the OTUs using a post hoc label-switching permutation test (26) (fig. S10). We found 20 genes that had significantly positive and 157 genes that had significantly negative associations with the T6SS. The genes positively associated with the T6SS primarily encode the T6SS itself (Fig. 3A). The genes negatively associated with the T6SS are mainly involved in metabolic processes, such as carbohydrate metabolism, metabolite transport, amino acid metabolism, and biosynthesis of secondary metabolites (Fig. 3A). This finding indicated that these metabolic functions are reduced in T6SS+ species. As an example for carbohydrate metabolism, we found that indeed T6SS+ Vibrio genomes contained significantly fewer enzymes to degrade alginate (1.54 ± 0.75 alginate lyases in T6SS+ and 6.03 ± 2.24 in T6SS- genomes) (Fig. 3B, fig. S11E), and the pan-genomic association study showed that alginate lyases of the PL7, PL12/PL17, and PL15/35 families were significantly negatively associated with the T6SS. The genome size and genome completeness were, however, not significantly different between T6SS+ and T6SS- OTUs (fig. S11A-C). Overall, the reduced repertoire of metabolic genes we observed, particularly for carbohydrate metabolism, in T6SS+ genomes is consistent with genomic adaptation toward acquisition of nutrients and macromolecules from external sources, rather than their biosynthesis from simple metabolites obtained from the decomposition of complex organic matter.
<figure><img src="image_5.png" /><figcaption>**Fig. 3.** *Vibrio* with T6SS have a reduced metabolic gene repertoire and bacteria with T6SS are prevalent in natural environments. (A) Counts of genes that are positively or negatively associated with the presence of the T6SS in 141 *Vibrio* species-level operational taxonomic units (OTUs), grouped into functional categories according to KEGG classification. A gene can be part of several KEGG classes. Functional categories with less than 3 significant genes are not shown. (B) Number of alginate lyase genes across *Vibrio* OTUs with and without the T6SS (n = 39, 82; Wilcoxon rank sum test, *p* = 1x10⁻⁵). (C) Genomes of the Ocean Microbiomics Database (OMD) (green-blue) placed onto the bacterial Genome Taxonomy Database (GTDB) backbone tree (gray) (modified from Fig. 2C in Paoli et al. (28)) with red squares indicating T6SS-encoding genomes. For visualization, the last 15% of the nodes were collapsed (color codes indicate the number of genomes collapsed). (D) Prevalence of T6SS-encoding OTUs in different ecosystems based on data from the genomic catalog of Earth's microbiomes (30).</figcaption></figure>

In the ocean, cell lysis induced by viruses is known to be an important contributor to carbon and nutrient cycling, in a phenomenon known as the ‘viral shunt’ (27). Cell lysis through bacterial antagonism may enhance this flux of carbon from living bacterial cells to lysis products. Hence, we screened for T6SS genes in the Ocean Microbiomics Database (OMD) (28), a consolidated collection of metagenome-assembled genomes from four major global ocean expeditions and two marine time series, as well as single-cell amplified genomes and marine microbial isolate genomes (fig. S12). In the combined 7,610 bacterial species-level
OTUs, we found T6SS in 281 OTUs i.e., in about 3.7%. In 229 of these OTUs more than half of the OTU members possessed a T6SS i.e., in about 3.0% (Fig. 3C). Apart from clades that are known to be rich in T6SS, like *Gammaproteobacteria* and *Bacteroidota*, we also found that *Planctomycetota* and *Rhodobacteraceae*, which are abundant in the coastal ocean (29), frequently encode T6SS. Overall, the prevalence of the T6SS in marine taxa from the global oceans suggests that T6SS-mediated nutrient foraging is common and may thus significantly influence nutrient cycling in the ocean.

A wider screen of natural environments showed that T6SS+ bacteria are common in diverse habitats. We screened for the presence of T6SS genes in a catalog of Earth's microbiomes (30), containing 45,599 species-level OTUs that include metagenome-assembled bacterial genomes from over 10,000 samples from diverse habitats. We found T6SS genes in 4–7% of OTUs in aquatic systems, in 9–11% of OTUs in terrestrial systems, and in 37% of OTUs in the rhizoplane (Fig. 3D). This is consistent with previous findings that suggest that T6SS+ bacteria are more common among particle-attached than among planktonic bacteria (13). Also, soils have the largest average number of cells per volume of any known ecological environment (31, 32), and the rhizosphere is considered to be the richest niche within this habitat (5, 33), which may promote contact-dependent interactions in this environment. In the rhizoplane, most bacteria are thought to be commensals, but plant pathogens were found that use the T6SS to dominate the root microbiota and increase their virulence against the host plant (5). In environments that are comparatively rich in organic biomass, like the digestive systems of animals and wastewater treatment plants, 6% and 7% of OTUs were T6SS+, respectively. These findings indicate that the T6SS is widespread across both nutrient-rich and nutrient-poor natural environments.

## Discussion

In this work, we found that contact-dependent antagonism can be used by bacteria to access nutrients from cell lysate, which can be rapidly assimilated at low metabolic cost. In contrast to hard-to-digest complex organic compounds prevalent in natural environments, such as polysaccharides, this source contains many readily available cellular building blocks, such as nucleotides and amino acids (25% and 57% of cell content, respectively) (34). Directly polymerizing these building blocks into functional cell components represents an energetic saving of more than 90% in comparison with first having to synthesize them, and also reduces proteomic cost (34). This mode of nutrient foraging is likely effective in microbial communities with a high frequency of cell–cell contact and limited diffusional loss of released nutrients, such as those in biofilms, soil aggregates, marine snow, the rhizosphere, and the intestinal mucus layer. It is less likely to bring an advantage in planktonic cells, but we have not assessed the efficiency of nutrient foraging in planktonic cells.

Nutrient foraging is an alternative function to the mediation of bacterial competition and other functions that have been ascribed to the T6SS, such as acquisition of genetic
material (12), defense (35), and metal scavenging (36). These functions are not mutually exclusive, but the genomic signatures of T6SS+ species in *Vibrio* match the expectations that arise from a role in nutrient foraging (24, 25). Nonetheless, it remains unknown whether antagonistic foraging in bacterial communities is the cause of this genomic adaptation, or whether other factors dominate. For example, some T6SS are active against eukaryotic hosts (9), which may contribute to an association between T6SS and reduced metabolic capacity in a host-associated lifestyle. The reduced repertoire of enzymes for carbohydrate degradation renders cells unable to grow on certain carbon sources e.g., alginate in the case of *V. anguillarum*. In these conditions, they are not in competition with other cells for the carbon source, but the possession of a T6SS enables them to acquire carbon from nearby cells, extending their ecological niche. Functional and genomic analyses of other genera are now necessary to determine whether this function is important in other T6SS-encoding taxa. Under a broad definition of predation i.e., killing cells for nutrient uptake, this interaction may be classified as facultative predation; however, there is no evidence that T6SS+ cells actively hunt target cells, and thus it does not meet stricter definitions of predation (37). In contrast to predation through ixotrophy, a recently described hunting strategy found in some filamentous T6SS+ bacterial species of the *Saprospiria* and *Cytophagia* classes with 'grappling hooks' to attach to prey cells (38), the T6SS+ cells studied here did not grasp target cells.

As the T6SS is found in diverse taxa and diverse environments, we expect inter-
bacterial cell lysis to also affect large-scale biogeochemical processes. Cell lysis releases both
labile dissolved organic matter and carbon-rich particulate organic matter (27). The pool of
labile substances provided by the lysate can be readily reused by bacteria (39) while the more
recalcitrant particulate organic matter may preferentially sink into deeper ocean layers and
thus contribute towards long-term carbon storage in the ocean, as proposed for the 'viral
shunt' (27). Our analyses suggest that there may be a parallel 'bacterial shunt' that
participates in marine biogeochemical cycling.

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## Acknowledgments

We thank current and previous members of the Schubert & Ackermann and Magnabosco labs and the PriME collaboration for helpful discussions, as well as Marco Gabrielli. We thank Hans Wildschutte for providing the antibiotic deletion of *Vibrio anguillarum* and Franziska Oschmann and Janis Fluri for the development of the segmentation and tracking tool *MIDAP*. We thank Knut Drescher and Niklas Netter for their advice on single cell segmentation of the T6SS cells. We thank Russell Naisbit for editorial comments and declare the use of ChatGPT-4 to inspire specific coding solutions.

## Funding

ETH Zurich and Eawag (MA and OS)

Simons Foundation Grants 542379FY22 and 542395FY22 as part of the Principles of Microbial Ecosystems (PriME) Collaboration (RS, OS, MA) and PriME Opportunity Grant from the PriME Collaboration (GD and JS)

ETH Zurich (CM)

ETH fellowship and a Marie Curie Actions for People COFUND program fellowship FEL-3716-1 (GD)

ETH Career SEED Grant SEED-14 18-1 (GD)

Gordon and Betty Moore Foundation Symbiosis in Aquatic Systems Initiative Investigator Award GBMF9197 (https://doi.org/10.37807/GBMF9197) (RS)

## Author contributions

Conceptualization: AS, MA, CM, GD

Methodology: AS, GD, FJP, KSL, JS, MB, LP

Investigation: AS, GD, FJP, KSL

Visualization: AS

Funding acquisition: MA, OS, GD, RS, JS
Project administration: AS

Supervision: MA, OS, CM, GD

Writing – original draft: AS

Writing – review & editing: AS, OS, MA, GD, CM, and all other co-authors

**Competing interests**

RS is also affiliated with PhAST inc., Zurich. KS is also affiliated with the Department of Biological Sciences and the Department of Biomedical Engineering, Ulsan National Institute of Science and Technology (UNIST), Ulsan, South Korea. Authors declare that they have no competing interests.

**Data and materials availability**

Code and data to reproduce the results and figures in this paper are available on ERIC Open (40). The stable isotope probing Raman microspectroscopy data is also deposited in the MicrobioRaman database (41).

**List of Supplementary Materials**

Materials and Methods

Figures S1 to S12

Tables S1 to S8

References (42-76)

Movies S1 to S6