Killing by Type VI secretion drives clonal phase separation and the evolution of cooperation

By nature of their small size, dense growth and frequent need for extracellular metabolism, microbes face persistent public goods dilemmas1–5. Spatial assortment can act as a general solution to social conflict by allowing extracellular goods to be utilized preferentially by productive genotypes1,6,7. Established mechanisms that generate microbial assortment depend on the availability of free space8–14; however, microbes often live in densely-packed environments, wherein these mechanisms are ineffective. Here, we describe a novel class of self-organized pattern formation that facilitates the development of spatial structure within densely-packed bacterial colonies. Contact-mediated killing through the Type VI secretion system (T6SS) drives high levels of assortment by precipitating phase separation, even in initially well-mixed populations that do not necessarily exhibit net growth. We examine these dynamics using three different classes of mathematical models and experiments with mutually antagonistic strains of Vibrio cholerae growing on solid media, and find that all appear to de-mix via the same ‘Model A’ universality class of order-disorder transition. We mathematically demonstrate that contact killing should favour the evolution of public goods cooperation, and empirically examine the relationship between T6SSs and potential cooperation through phylogenetic analysis. Across 26 genera of Proteobacteria and Bacteroidetes, the proportion of a strain’s genome that codes for potentially-exploitable secreted proteins increases significantly with boththe number of Type 6 secretion systems and the number of T6SS effectors that it possesses. This work demonstrates how antagonistic traits—likely evolved for the purpose of killing competitors—can indirectlylead to the evolution of cooperation by driving genetic phase separation.


Summary
By nature of their small size, dense growth and frequent need for extracellular metabolism, microbes face persistent public goods dilemmas [1][2][3][4][5] . Spatial assortment can act as a general solution to social conflict by allowing extracellular goods to be utilized preferentially by productive genotypes 1,6,7 .
Established mechanisms that generate microbial assortment depend on the availability of free space 8-14 ; however, microbes often live in densely-packed environments, wherein these mechanisms are ineffective. Here, we describe a novel class of self-organized pattern formation that facilitates the development of spatial structure within densely-packed bacterial colonies. Contact-mediated killing through the Type VI secretion system (T6SS) drives high levels of assortment by precipitating phase separation, even in initially well-mixed populations that do not necessarily exhibit net growth. We examine these dynamics using three different classes of mathematical models and experiments with mutually antagonistic strains of Vibrio cholerae growing on solid media, and find that all appear to demix via the same 'Model A' universality class of order-disorder transition. We mathematically demonstrate that contact killing should favour the evolution of public goods cooperation, and empirically examine the relationship between T6SSs and potential cooperation through phylogenetic analysis. Across 26 genera of Proteobacteria and Bacteroidetes, the proportion of a strain's genome that codes for potentially-exploitable secreted proteins increases significantly with both the number of Type 6 secretion systems and the number of T6SS effectors that it possesses. This work demonstrates how antagonistic traits-likely evolved for the purpose of killing competitors-can indirectly lead to the evolution of cooperation by driving genetic phase separation.

Results and Discussion
Microbes are fundamentally social organisms. They often live in dense, surface-attached communities, and participate in a range of social behaviors mediated through the production and consumption of extracellular proteins and metabolites. Paradigmatic examples include the cooperative production of digestive enzymes 15 , metal chelators 16 , signaling molecules 15 , and the structural components of biofilms 17 . Many of these extracellular compounds are susceptible to social exploitation, in which non-producing 'cheats' gain an evolutionary advantage. If unchecked, this social exploitation can lead to the extinction of cooperative genotypes 18,19 . While it is widely recognized that the spatial segregation of cooperative microbes away from cheats can solve this cooperative dilemma 1,5,19 , relatively little work has investigated mechanisms that can generate genetic segregation within initially mixed communities. Using a bacterial model system 20 and mathematical modeling, we examine the biophysical basis of novel ecological structuring created by contact-mediated killing through the Type VI secretion system (T6SS). The T6SS is a potent mechanism of intermicrobial aggression, allowing bacteria to deliver lethal doses of effector proteins to adjacent competitors, while leaving clonemates with identical protective immunity proteins unscathed 21,22 .
Our system illustrates the profound effect of T6SS-mediated killing on emergent spatial patterning of a surface attached population. Mathematical modeling suggests that an initially wellmixed population of mutual killers should rapidly undergo phase separation due to 'selfish herd' dynamics 23 , as the cells within genetically-uniform groups no longer risk T6SS-mediated death. Indeed, we observe rapid phase separation in three distinct classes of models, all starting with a randomly seeded population on a 2D lattice ( To determine whether our models and experiments undergo the same type of order-disorder transition, we quantitatively examined the dynamics of phase separation in each. We first computed the Fourier-transformed structure factor, S(q); to facilitate comparisons between models and experiments, where L is the size of a unit cell. The peak in S * (q) identifies the most common characteristic length scale (the inverse of q) of clonal groups, and the height of the peak is related to how often it occurs in the lattice (i.e. the strength of patterning at that length scale). At early timesteps (Fig. 2a), or for non-killing controls (Fig. 2b), S * (q) is relatively flat, as expected for a well-mixed population lacking a characteristic length scale. T6SS-mediated killing causes a peak to appear in S * (q), which grows in height and moves to smaller values of q (longer length scales) as the population grows increasingly structured. This progression of S * (q) is a hallmark of phase separation 26 .
The location of the first peak in S * (q), denoted q max , scales as ∝ 1 √ ⁄ , while the height of the peak scales 27 as * ( ) ∝ √ . It is ambiguous how to relate simulation time to experimental time; instead, we plot S * (q max ) versus q max . Remarkably, all models (IBM, PDE and Ising) and experiments fall on the same line (S * (q max )~1 / q max ) (Fig. 2c), a relationship consistent with the "Model A" order-disorder phase separation process 28  To provide biological context for this process of phase separation, we calculated clonal assortment (r), for the IBM (Fig. 2e) and the Vibrio experiments (Fig. 2f). T6SS-mediated killing resulted in the creation of highly structured populations with high assortment over long length scales (Fig. 2e&f), which can protect diffusible public goods from consumption by competing strains 30,31 . To explore the effect of T6SS-mediated killing on the evolutionary stability of public goods cooperation, we introduced a diffusible cooperative good into our PDE model. We considered two competing strains: a cooperator that secretes an exoproduct into its environment at an individual cost, and a non-producing cheat that, all else equal, grows faster than the cooperator as it does not pay the cost of production. In this model, cellular growth rates for both strains depend on the local concentration of the diffusible exoproduct. We find that T6SS-mediated killing protects cooperation in two different ways. In a non-spatial (i.e.,  (Fig. 4b, d, Table S2) and T6SS effector proteins (Fig. 4c, e, Table S2) present. These results are also robust in univariate analyses (Tables S3 and   S4) and to the inclusion of genome size as a predictor (Table S5). As our analyses include many closely related strains (e.g., many Helicobacter pylori, Fig. 4a), most (91%) of the variance in secretome size is explained by the phylogenetic relationships among strains. Nonetheless, the number of T6 secretion systems and T6SS effectors are important predictors of secretome size, explaining 8% of the total, and 90% of the non-phylogenetic variance in secretome size. While, as with any phylogenetic analysis, alternative hypotheses cannot be ruled out entirely, these results strongly suggest that T6SS-mediated killing creates conditions that favour exoproduct evolution across a broad diversity of bacterial taxa.
Phase separation is well-known to drive pattern formation in biology 14 , but has mainly been investigated using either Turing activator-inhibitor feedbacks 33,34 , or positive density-dependent movement, described by the Cahn-Hilliard equation 14,[35][36][37] . In this paper we describe a third general mechanism of self-organized pattern formation: targeted killing of non-kin competitors. This drives a 'Model A' phase separation; the kinetics of this coarsening process-described by the Allen-Cahn equation-only depend on a few cellular details. While we explore this process in bacteria, it is probably more general, applying to other organisms that kill adjacent non-kin (e.g., allelopathy in plants 38 and animals 39 ).
In recent years, there has been a growing appreciation that many microbial behaviors requiring extracellular metabolism are susceptible to social exploitation. Here we show how simple cell-cell aggression can, as a consequence, create a structured population favourable to cooperation. Because T6SSs are common (found in ~25% of gram negative bacteria 21 ), and microbes often live in dense communities, phase-separation driven by contact-mediated killing may play a fundamental role in defining the genetic composition and ecosystem-level functionality of microbial communities globally.  . We also examine the creation of spatial structure by calculating a biological metric, assortment (r), through time in the IBM (e) and after 24 h in experiments (f). Mutual killers were grown at 30°C (red), 25°C (blue) and 17°C (green). Defective killers were grown at 30°C (purple), 25°C (teal) and 17°C (orange). Plotted is the mean assortment of ≥ 3 replicate populations ± 95% confidence intervals. In the absence of T6SS-mediated killing, cooperation is not favoured in either a well-mixed environment (a) or a spatially-defined environment (b). In a non-spatial environment with killing via T6SS, cooperators can be protected from cheats when common owing to their advantage in antagonistic interactions, but cannot invade from rarity (c). In contrast, the high assortment created by phase separation allows cooperators to invade from rarity and spread to fixation (d). The spatial organization of cooperators (blue) and cheats (red) during competition is shown in (e). Panels correspond to the time-points marked by circles in (d).  (Tables S3 and S4). Posterior distributions of the effects of the numbers of T6SS (d) and T6SS effectors (e) on secretome size from the multivariate BPMM (Table S2). 95% credible intervals of the estimates are shaded. Plot of observed against predicted secretome size from the multivariate BPMM (f), including effects of the number of T6SS, number of T6SS effectors and phylogeny. The line represents a 1:1 mapping.