Biology subjects

Smith, W. P. J.

Publications and source records attributed to Smith, W. P. J..

6 recordsLinked to original sources

bioRxiv2026

Evolved resistance against the type 6 secretion system is toxin specific

Across ecosystems, microbes face attacks by competitors armed with lethal weapons, including toxin-injecting Type 6 Secretion Systems (T6SSs). This in turn selects for T6SS resistance, reducing the future effectiveness of T6SS weaponry in competition. However, we have only a limited understanding of how resistance to T6SS attacks evolves de novo. A key challenge is that T6SS-armed bacteria are highly diverse in the number and type of toxins they inject, spanning multiple distinct modes of action. Do functionally distinct T6SS toxins select for different mechanisms of resistance? To address this, we combine genomics, resistance assays and fluorescence microscopy, to characterise evolved resistances against common T6SS toxin classes across spatial scales. We discovered that amidase and lipase toxins select for mutations in distinct sets of genes, resulting in toxin-specific resistance phenotypes and fitness costs. We also discovered resistance trade-offs: lipase-evolved E. coli became less vulnerable to lipase membrane damage, at the cost of increased susceptibility to lysis by amidase toxins. Finally, using single-gene knockout mutants from the Keio collection we confirmed that specific genes not previously linked to T6SS resistance, including inner membrane transporters, osmo-sensing systems and stress response pathways, conferred resistance to specific toxins while generating sensitivity to others. The specificity of resistance, and associated trade-offs observed, are likely to constrain de novo evolution of resistance against T6SS attackers armed with multiple functionally distinct toxins, helping to explain why T6SS systems are so widespread in nature.

microbiology↗

bioRxiv2025

Type VI secretion system activity at lethal antibiotic concentrations leads to overestimation of weapon potency

Competition assays are a mainstay of modern microbiology, offering a simple and cost-effective means to quantify microbe-microbe interactions in vitro. Here we demonstrate a key weakness of this method that arises when competing microbes interact via toxins, such as those secreted via the type VI secretion system (T6SS). Timelapse microscopy reveals T6SS-armed Acinteobacter baylyi bacteria can maintain lethal T6SS activity against E. coli target cells, even under selective conditions intended to eliminate A. baylyi. Further, this residual killing creates a density- and T6SS-dependent bias in the apparent recovery of E. coli, leading to a misreporting of competition outcomes especially where target survival is low. We also show that incubating A. baylyi / E. coli co-cultures in liquid antibiotic prior to selective plating can substantially correct this bias. Our findings demonstrate the need for caution when using selective plating as part of T6SS competition assays, or assays involving other toxin-producing bacteria.

microbiology↗

bioRxiv2024

Multiplicity of Type 6 Secretion System toxins limits the evolution of resistance

The bacterial Type 6 Secretion System (T6SS) is a toxin-injecting nanoweapon that mediates competition in plant- and animal-associated microbial communities. Bacteria can evolve de novo resistance against T6SS attacks, but resistance is far from universal in natural communities, suggesting key features of T6SS weaponry may act to limit its evolution. Here, we combine eco-evolutionary modelling and experimental evolution to examine how toxin type and multiplicity in Acinetobacter baylyi attackers shape resistance evolution in susceptible Escherichia coli competitors. In both our models and experiments, we find that combinations of multiple distinct toxins limit resistance evolution by creating genetic bottlenecks, driving resistant lineages extinct before they can reach high frequency. We also show that, paradoxically, single-toxin attackers often drive the evolution of cross-resistance, protecting bacteria against unfamiliar toxin combinations, even though such evolutionary pathways were inaccessible against multi-toxin attackers. Our findings indicate that, comparable to antimicrobial and anticancer combination therapies, multi-toxin T6SS arsenals function to limit resistance evolution in competing microbes. This helps us to understand why T6SSs remain widespread and effective weapons in microbial communities, and why many bacteria T6SS-armed encode functionally diverse anti-competitor toxins. SignificanceToxin secretion systems, such as T6SSs, are widely used by bacteria to inhibit competing microorganisms. Here, we show that the secretion of multiple toxins in combination can suppress the evolution of resistance to the T6SS, rationalising its continued widespread use across diverse communities. Our work shows that principles of combination therapy--well known in the context of antimicrobial, antiviral and anticancer therapies--also apply in the context of microbial warfare, helping to explain why many bacteria maintain diverse T6SS toxin arsenals. Resistance suppression is also a technologically useful property of T6SS toxin cocktails, which could be harnessed as part of future biocontrol or biotherapeutic applications, using live T6SS-armed bacteria to limit the growth of problem microorganisms.

microbiology↗

bioRxiv2022

Bows and swords: why bacteria carry short and long-range weapons

Bacteria possess a diverse range of mechanisms for inhibiting competitors, including bacteriocins, tailocins, the type VI secretion system, and contact-dependent inhibition. Why bacteria have evolved such a wide array of weapon systems remains a mystery. Here we develop an agent-based model to compare short-range weapons that require cell-cell contact, with long-range weapons that rely on diffusion. Our models predict that contact weapons are useful when an attacking strain is outnumbered, facilitating invasion and establishment. By contrast, ranged weapons tend to only be effective when attackers are abundant. We test our predictions with the opportunistic pathogen Pseudomonas aeruginosa, which naturally carries multiple weapons, including contact-dependent inhibition (CDI) and diffusing tailocins. As predicted, short-range CDI functions better at low frequency, while long-range tailocins require high frequency and cell density to function effectively. Head-to-head competitions between the two weapon types further support our predictions: a tailocin attacker only defeats CDI when it is numerically dominant, but then we find it can be devastating. Finally, we show that the two weapons work well together when one strain employs both. We conclude that short and long-range weapons serve different functions and allow bacteria to fight both as individuals and as a group.

microbiology↗

bioRxiv2022

Collective protection against the type VI secretion system in bacteria

Bacteria commonly face attacks from other strains using the type VI secretion system (T6SS), a molecular speargun that stabs and intoxicates competitors. Here we show how bacteria can work together to collectively defend themselves against these attacks. This project began with an outreach activity: while developing an online computer game of bacterial warfare, we noticed that one strategist ( Slimy) that made extracellular polymeric substances (EPS) was able to resist attacks from another strategist that employed the T6SS ( Stabby). This observation motivated us to model this scenario more formally, using dedicated agent-based simulations. The model predicts that EPS production can serve as a collective defence mechanism, which protects both producing cells and neighbouring cells that do not make EPS. We then tested our model with a synthetic community that contains a T6SS-wielding attacker (Acinetobacter baylyi), and two T6SS-sensitive target strains (Escherichia coli) that either secrete EPS, or not. As predicted by our modelling, we find that the production of EPS leads to collective protection against T6SS attacks, where EPS producers protect each other and nearby non-producers. We identify two processes that explain this protection: EPS sharing between cells and a second general mechanism whereby groups of resistant cells shield susceptible cells ( flank protection). Our work shows how EPS-producing bacteria can work together to defend themselves from the type VI secretion system.

microbiology↗

bioRxiv2020

Droplet printing reveals the importance of micron-scale structure for bacterial ecology

Bacteria often live in diverse communities where the spatial arrangement of strains and species is considered critical for their ecology, including whether strains can coexist, which are ecologically dominant, and how productive they are as a community1,2. However, a test of the importance of spatial structure requires manipulation at the fine scales at which this structure naturally occurs3-8. Here we develop a droplet-based printing method to arrange different bacterial genotypes across a sub-millimetre array. We use this to test the importance of fine-scale spatial structure by printing strains of the gut bacterium Escherichia coli that naturally compete with one another using protein toxins9,10. This reveals that the spatial arrangement of bacterial genotypes is important for ecological outcomes. Toxin-producing strains largely eliminate susceptible non-producers when genotypes are well-mixed. However, printing strains side-by-side creates an ecological refuge such that susceptible strains can coexist with toxin producers, even to the extent that a susceptible strain outnumbers the toxin producer. Head-to-head competitions between toxin producers also reveals strong effects, where spatial structure can make the difference between one strain winning and mutual destruction. Finally, we print different potential barriers between two competing strains to understand why space is so important. This reveals the importance of processes that limit the free diffusion of molecules. Specifically, we show that cells closest to a toxin producer bind to and capture toxin molecules, which creates a refuge for their clonemates. Our work provides a new method to generate customised bacterial communities with defined spatial distributions, and reveals that micron-scale changes in these distributions can drive major shifts in their ecology.

microbiology↗