Biology subjects

Westra, E. R.

Publications and source records attributed to Westra, E. R..

At least 19 recordsLinked to original sources

bioRxiv2026

Maintenance of restriction-modification system diversity creates pan-immunity

Bacterial antiviral defense systems are highly diverse and unevenly distributed across populations, such that different strains carry distinct immune repertoires that together form a pan-immune system. Restriction-modification (RM) systems are the most abundant bacterial defense systems and exemplify this architecture, exhibiting remarkable diversity in structure, function, specificity, and efficacy. How and why such diversity is maintained, why weaker systems are not outcompeted, and whether diversity provides benefits beyond protection of individual cells remain unclear. Here, we experimentally tracked bacteria and phages across populations differing in RM diversity. We find no consistent evidence of a trade-off between defense strength and host fitness. Instead, we identify a trade-off between defense strength and horizontal transferability: stronger RM systems are less able to spread between strains, likely because factors that increase defense strength also increase toxicity risk upon transfer to unmethylated hosts. These weaker yet more mobile systems may therefore act as more accessible pan-immune components that bacterial populations can acquire in response to shifting parasite pressures. Furthermore, we show RM systems experience negative frequency-dependent selection, such that common specificities are more readily overcome by phages while rare ones remain protective, causing the benefits of potent systems to erode as they spread. Together, these dynamics maintain RM diversity and explain why weaker systems persist alongside stronger ones. Finally, we show that population-level RM diversity generates collective protection by constraining the emergence of RM-escaping phages and limiting the invasion of bacterial surface mutants that avoid phage adsorption, providing experimental support for the pan-immune model.

microbiology↗

bioRxiv2026

Loss of restriction-modification methyltransferases drives persistence to fluoroquinolones in Pseudomonas aeruginosa

The resurgence of phage therapy has renewed interest in the interplay between phage resistance and antibiotic susceptibility and has stimulated research in the emergent field of non-canonical cellular functions carried out by defence systems. Yet it remains largely unknown whether intracellular antiphage defence systems influence bacterial physiology, resistance or persistence to antibiotics. Here we discovered that besides its canonical antiphage defence function, the type I restriction modification (RM) system profoundly affects the physiology of the opportunistic pathogen Pseudomonas aeruginosa. Deletion of the type I RM methyltransferase HsdM reduces the size of the bacterial nucleoid and delays DNA replication initiation and exit from lag phase in P. aeruginosa PAO1. Crucially, P. aeruginosa strains isolated from patients with cystic fibrosis (CF) and lacking the RM type I system also display slower growth compared to strains isolated from other sites of infections and encoding this system. Deletion of HsdM selectively increases the levels of persisters that survive treatment with fluoroquinolones by displaying enhanced SOS response but without acquiring resistance. Importantly, we measured elevated persistence to fluoroquinolones also in P. aeruginosa CF isolates lacking the type I RM system, providing a functional link between RM systems, slow growth and persistence to fluoroquinolones. Together these findings open a new way of approaching bacterial susceptibility to antibiotics, bringing antiphage defence systems in a forward-facing position in this field.

microbiology↗

bioRxiv2026

Dual ATPase-activated DNA hemimethylation and cleavage by the MANTIS defence system

Bacterial defence systems are diverse and form major barriers to horizontal gene transfer and bacteriophage (phage) infection. Though restriction-modification systems were the first such defences discovered and remain the most widespread, the basis of self/non-self discrimination in many newly identified methylation-based systems remains unclear. Here, we define the mechanism of a widely distributed BREX-related defence family, previously termed Gao_RL, which we rename MANTIS (Methyltransferase, ATPases and Nuclease Targeting Inverted-repeat Sequences). Using phage infection and plasmid transformation assays in Pseudomonas aeruginosa, biochemical reconstitution, and structural biology, we show that MANTIS establishes self-identity through adenine hemimethylation of short asymmetric DNA sequences. Unexpectedly, a AAA+ ATPase (MtsA) and a co-regulator (MtsB) are both required to recruit and activate the methyltransferase MtsC, revealing a previously unknown ATP-dependent mode of epigenetic modification. DNA recognition by the MtsABC complex then triggers restriction by a dimeric helicase-nuclease effector (MtsD), with activity dependent on target-site architecture: inverted pairs of unmodified sites support robust restriction, whereas direct repeats or single sites are restricted less efficiently. Consistent with this model, virulent phages show strand-biased depletion of MANTIS target sites, indicating evolutionary pressure to evade restriction. Together, these findings reveal how dual ATPases couple self-modification to non-self restriction and provide a framework for understanding the expanding landscape of methylation-based defence systems.

molecular biology↗

bioRxiv2026

Engineering the Pseudomonas aeruginosa virus-isolation host viPAO1 reveals how host genetic barriers shape recoverable phage diversity

Cultured phage collections provide essential access to viral biology, but they capture only a small and biased fraction of natural phage diversity. Key barriers to diverse phage isolation are likely to include the prophages and defence systems present in the genomes of isolation hosts. Here, we show that removal of these host genetic barriers expands recoverable phage diversity in Pseudomonas aeruginosa. We constructed a series of progressively more permissive hosts derived from MPAO1 by sequentially deleting intracellular defence systems and resident prophages, culminating in viPAO1 (virus-isolation PAO1). Sequential removal of these barriers increased phage susceptibility, with Gabija and type I restriction-modification acting as major barriers to phage infection. In comparative isolation experiments, removal of intracellular barriers produced the largest increase in phage recovery, while additional receptor modification altered the composition of recovered phage subsets. viPAO1 also substantially increased recovery of phage contigs from environmental samples. Scaling this approach across 1090 clinical isolates enabled recovery of a curated collection of 69 individually purified temperate P. aeruginosa phages spanning 24 predicted genera, including 15 putative novel genera. Together, these findings demonstrate that removal of host genetic barriers to infection enhances recoverable phage diversity and establish permissive host engineering as a practical strategy for uncovering under-sampled regions of cultured phage diversity.

microbiology↗

bioRxiv2026

Comprehensive profiling of infant gut virome assembly reveals associations with eczema and wheeze

Infancy is a critical developmental window during which the gut ecosystem assembles and helps train the immune system, thereby setting trajectories for lifelong health. Bacteria and viruses are equally numerous in this early ecosystem, yet the gut viromes composition, dynamics, and health relevance remain poorly understood. Here, we show that the infant gut virome is diverse, dynamic, and linked to health outcomes. We performed comprehensive virome profiling of 1,110 longitudinal fecal samples from 314 mother-infant pairs from the Dutch birth cohort Lifelines NEXT using both virus-like particle enrichment (VLP) and total metagenomic sequencing (MGS). We find only 18.9% compositional overlap between the VLP- and MGS-metaviromes, with VLP recovering the active virome and most novel species and MGS predominantly capturing temperate phages. By combining both methods, we identified 8,348 novel virus species spanning diverse hosts, from bacteria to humans, and all major viral genome types (dsDNA, ssDNA, and RNA). We find that bacteriophages frequently encode metabolic functions, including genes related to B vitamin metabolism. We further observe that the development of the infant gut virome is shaped by both host factors, including delivery mode and feeding practices, and continuous switching of temperate phage lifecycles. Notably, the relative abundance of induced temperate phages is also associated with eczema development within the first year of life. Together, these findings establish the infant gut virome as a dynamic and clinically relevant component of early-life microbial development and highlight how comprehensive dual-method profiling is a necessary framework for future virome research.

microbiology↗

bioRxiv2025

The ambivalent effect of spatial structure on the spread of cooperative anti-CRISPR phages

Some phages have evolved the ability to cooperate to evade the immunity triggered by their bacterial host. A first exposure to the phage may weaken the host defences and allow later infections to be successful. Because this cooperation requires sequential infections, the phage can invade the host population only if its initial density is sufficiently high in a well-mixed environment. However, most natural bacterial populations are spatially structured. Could spatial structure create more favourable conditions for viral spread? Here we study the effect of spatial structure on the dynamics of cooperative anti-CRISPR (Acr) phages spreading in a population of CRISPR-Cas resistant Pseudomonas aeruginosa bacteria. We show experimentally that spatial structure does not always promote the spread of Acr-phages. In particular, the effect of spatial structure is modulated by the efficacy of the bacterial hosts CRISPR-Cas resistance and by the efficacy of the phage Acr protein. These results are discussed in the light of a mathematical model we developed to describe the spread of the phage. The model allows us to understand the ambivalent effects of spatial structure via its effects on the reproduction and on the persistence of the phage. More generally, we find that spatial structure can have opposite effects on the epidemiological dynamics of the phage, depending on the properties of the Acr protein encoded by the phage. This joint experimental and theoretical work yields a deeper understanding of the spatial dynamics of cooperative strategies in phages.

microbiology↗

bioRxiv2025

Defence systems drive accessory genome interactions in Pseudomonas aeruginosa.

Bacterial genomes represent dynamic ecological systems in which mutational selection and dynamic accessory genome element complements drive evolution. Emerging evidence suggests that bacterial defence systems, which protect against phages and other mobile genetic elements, interact through cooperative, competitive, and antagonistic relationships. These interactions can influence horizontal gene transfer, shape phage susceptibility, and the diversification of genome composition across environments. Recent ecological studies describe non-random co-occurrence and avoidance of defence systems, suggesting that they may emerge from ecological and evolutionary interactions, rather than by chance. This necessitates exploring whether these patterns reflect functional compatibility, shared selection pressures imposed by ecological niche, or if they merely arise from co-localisations of convenience. Understanding these patterns is key to elucidating how the accessory genome evolves and how defence systems constrain or facilitate genome plasticity. To investigate these patterns, in their genomic context, and provide a resource for future investigations, we analysed the distributions of defence systems and other accessory genome elements in a recently curated global dataset of 2,940 Pseudomonas aeruginosa genomes. Genomic defence system content varied by ecological niche, with higher numbers per genome in non-cystic fibrosis derived isolates (average n=7.9) compared to cystic fibrosis-derived isolates (average n=6.5). We identified multiple associations (n=426) and dissociations (n=50) among defence systems, and among other accessory genome elements, many of which had a plausible biological explanation. We also quantitated the relative interactions among accessory genome elements which revealed that defence systems and anti-defence systems engage in the most accessory genome interactions, compared with e.g. antimicrobial resistance genes, plasmids, phages, suggesting that systems are a major driving force in the ecological dynamics of the bacterial accessory genome. Together, these patterns provide new insights into the evolutionary forces shaping bacterial genomes, provide a valuable resource of quantitated interactions in a highly curated Pseudomonas aeruginosa dataset, and establish a framework for future mechanistic and ecological investigations of defence system interactions.

microbiology↗

bioRxiv2025

Phage susceptibility to a minimal, modular synthetic CRISPR-Cas system in Pseudomonas aeruginosa is nutrient dependent

CRISPR-Cas systems can provide adaptive, heritable immunity to their prokaryotic hosts against invading genetic material such as phages. It is clear that the importance of acquiring CRISPR-Cas immunity to anti-phage defence varies across environments, but it is less clear if and how this varies across different phages. To explore this, we created a synthetic, modular version of the type I-F CRISPR-Cas system of Pseudomonas aeruginosa. We used this synthetic system to test CRISPR-Cas interference against a panel of 13 diverse phages using engineered phage-targeting spacers. We observed complete protection against eight of these phages, both lytic and lysogenic and with a range of infectivity profiles. However, for two phages CRISPR-Cas interference was only partially protective in high nutrient conditions, yet completely protective in low nutrient conditions. This work demonstrates that nutrient conditions modulate the strength of CRISPR-Cas immunity and highlights the importance of environmental conditions when screening defence systems for their efficacy against various phages.

microbiology↗

bioRxiv2024

Various plasmid strategies limit the effect of bacterial Restriction-Modification systems against conjugation.

In bacteria, genes conferring antibiotic resistance are mostly carried on conjugative plasmids, mobile genetic elements which spread horizontally between bacterial hosts. Bacteria carry defence systems which defend them against genetic parasites, but how effective these are against plasmid conjugation is poorly understood. Here, we study to what extent Restriction-Modification (RM) systems - by far the most prevalent bacterial defence systems - act as a barrier against plasmids. Using 10 different RM systems and 13 natural plasmids conferring antibiotic resistance in Escherichia coli, we uncovered variation in defence efficiency ranging from none to 105-fold protection. Further analysis revealed genetic features of plasmids that explain the observed variation in defence levels. First, the number of RM recognition sites present on the plasmids generally correlates with defence levels, with higher numbers of sites being associated with stronger defence. Secondly, some plasmids encode methylases that protect against restriction activity. Finally, we show that a high number of plasmids in our collection encode anti-restriction genes that provide protection against several types of RM systems. Overall, our results show that it is common for plasmids to encode anti-RM strategies, and that, as a consequence, RM systems form only a weak barrier for plasmid transfer by conjugation.

microbiology↗

bioRxiv2023

The impact of phage and phage resistance on microbial community dynamics

Where there are bacteria, there will be bacteriophages. These viruses are known to be important players in shaping the wider microbial community in which they are embedded, with potential implications for human health. On the other hand, bacteria possess a range of distinct immune mechanisms that provide protection against bacteriophages, including the mutation or complete loss of the phage receptor, and CRISPR-Cas adaptive immunity. Yet little is known about how interactions between phages and these different phage resistance mechanisms affect the wider microbial community in which they are embedded. Here, we conducted a 10-day, fully factorial evolution experiment to examine how phage impact the structure and dynamics of an artificial four-species bacterial community that includes either Pseudomonas aeruginosa wild type or an isogenic mutant unable to evolve phage resistance through CRISPR-Cas. Our results show that the microbial community structure is drastically altered by the addition of phage, with Acinetobacter baumannii becoming the dominant species and P. aeruginosa being driven nearly extinct, whereas P. aeruginosa outcompetes the other species in the absence of phage. Moreover, we find that a P. aeruginosa strain with the ability to evolve CRISPR-based resistance generally does better when in the presence of A. baumannii, but that this benefit is largely lost over time as phage is driven extinct. Combined, our data highlight how phage-targeting a dominant species allows for the competitive release of the strongest competitor whilst also contributing to community diversity maintenance and potentially preventing the reinvasion of the target species, and underline the importance of mapping community composition before therapeutically applying phage.

microbiology↗

bioRxiv2023

Eco-evolutionary feedbacks shape the evolution of constitutive and inducible defences

Organisms have evolved a range of constitutive (always active) and inducible (elicited by parasites) defence mechanisms, but we have limited understanding of what drives the evolution of these orthogonal defence strategies. Bacteria and their phages offer a tractable system to study this: bacteria can acquire constitutive resistance by mutation of the phage receptor (surface mutation, sm) or induced resistance through their CRISPR-Cas adaptive immune system. Using a combination of theory and experiments we demonstrate that the mechanism that establishes first has a strong advantage because it weakens selection for the alternative resistance mechanism. As a consequence, ecological factors that alter the relative frequencies at which the different resistances are acquired have a strong and lasting impact: high growth conditions promote the evolution of sm resistance by increasing the influx of receptor mutation events during the early stages of the epidemic, whereas a high infection risk during this stage of the epidemic promotes the evolution of CRISPR immunity, since it fuels the (infection-dependent) acquisition of CRISPR immunity. This work highlights the strong and lasting impact of the transient evolutionary dynamics during the early stages of an epidemic on the long-term evolution of constitutive and induced defences, which may be leveraged to manipulate phage resistance evolution in clinical and applied settings.

evolutionary biology↗

bioRxiv2022

Antibiotics that affect translation can antagonize phage infectivity by interfering with the deployment of counter-defences.

It is becoming increasingly clear that antibiotics can both positively and negatively impact the infectivity of bacteriophages (phage), but the underlying mechanisms often remain unclear. Here we demonstrate that antibiotics that target the protein translation machinery can fundamentally alter the outcome of bacteria-phage interactions by interfering with the production of phage-encoded counter-defence proteins. Specifically, using Pseudomonas aeruginosa PA14 and phage DMS3vir as a model, we show that bacteria with CRISPR-Cas immune systems have elevated levels of immunity against phage that encode anti-CRISPR (acr) genes when translation inhibitors are present in the environment. CRISPR-Cas are highly prevalent defence systems that enable bacteria to detect and destroy phage genomes in a sequence-specific manner. In response, many phages encode acr genes that are expressed immediately following infection to inhibit key steps of the CRISPR-Cas immune response. Our data show that while phage carrying acr genes can amplify efficiently on bacteria with CRISPR-Cas immune systems in the absence of antibiotics, the presence of antibiotics that act on protein translation prevents phage amplification, while protecting bacteria from lysis. These results help to understand how antibiotics-phage synergy and antagonism depend on the molecular interactions that define phage infectivity and host immunity.

microbiology↗

bioRxiv2022

Interspecific competition can drive the loss of conjugative plasmids from a focal species in a microbial community

Plasmids are key disseminators of antimicrobial resistance genes and virulence factors, and it is therefore critical to predict and reduce plasmid spread within microbial communities. The cost of plasmid carriage is a key metric that can be used to predict plasmids ecological fate, and it is unclear whether plasmid costs are affected by growth partners in a microbial community. We carried out competition experiments and tracked plasmid maintenance using a synthetic and stable 5-species community and a broad host-range plasmid as a model. We report that both the cost of plasmid carriage and its long-term maintenance in a focal strain depended on the presence of competitors, and that these interactions were species-specific. Addition of growth partners increased the plasmid cost to a focal strain, and accordingly plasmid loss from the focal species occurred over a shorter time frame in these species combinations. We propose that the destabilising effect of interspecific competition on plasmid maintenance may be leveraged in clinical and natural environments to cure plasmids from focal strains.

microbiology↗

bioRxiv2021

High viral abundance and low diversity are associated with increased CRISPR-Cas prevalence across microbial ecosystems

CRISPR-Cas are adaptive immune systems that protect their hosts against viruses and other parasitic mobile genetic elements. Consequently, selection from viruses and other genetic parasites is often assumed to drive the acquisition and maintenance of these immune systems in nature, but this remains untested. Here, we analyse the abundance of CRISPR arrays in natural environments using metagenomic datasets from 332 terrestrial, aquatic and host-associated ecosystems. For each metagenome we quantified viral abundance and levels of viral community diversity to test whether these variables can explain variation in CRISPR-Cas abundance across ecosystems. We find a strong positive correlation between CRISPR-Cas abundance and viral abundance. In addition, when controlling for differences in viral abundance, we found that the CRISPR-Cas systems are more abundant when viral diversity is low. We also found differences in relative CRISPR-Cas abundance among environments, with environmental classification explaining [~]24% of variation in CRISPR-Cas abundance. However, the correlations with viral abundance and diversity are broadly consistent across diverse natural environments. These results indicate that viral abundance and diversity are major ecological factors that drive the selection and maintenance of CRISPR-Cas in microbial ecosystems. Significance statementNumerous studies demonstrate that CRISPR-Cas immune systems can provide defence against bacteriophage and archaeal viruses, yet little is known about the ecological conditions where CRISPR-Cas immunity is favoured. Moreover, our knowledge is largely confined to laboratory studies and it is unknown if viruses are a key selective driver of CRISPR-Cas in nature. Using metagenomic data from diverse environments we find that both viral abundance and the abundance of CRISPR-Cas immune systems correlate positively across most environments. Furthermore, CRISPR-Cas systems are more prevalent when viral diversity is low. These results extend previous theoretical work by demonstrating that viruses are a key driver of selection of CRISPR-Cas immune systems across many natural ecosystems.

microbiology↗

bioRxiv2021

Bacteriostatic antibiotics promote the evolution of CRISPR-Cas immunity

Phage therapy can be used in combination with antibiotics to combat infections with bacterial pathogens1-3. However, bacteria can rapidly evolve phage resistance via receptor mutation, or using their CRISPR-Cas adaptive immune systems4, which insert short phage-derived sequences into CRISPR loci in the bacterial genome5 to guide sequence-specific cleavage of cognate sequences6. Unlike CRISPR-Cas immunity, mutation of the phage receptor leads to attenuated virulence when the opportunistic pathogen Pseudomonas aeruginosa is infected with its phage DMS3vir7, which underscores the need to predict how phage resistance evolves under clinically relevant conditions. Here, using eight antibiotics with various modes of action, we show that bacteriostatic antibiotics (which inhibit cell growth without killing) specifically promote evolution of CRISPR-Cas immunity in P. aeruginosa by slowing down phage development and providing more time for cells to acquire phage-derived sequences and mount an immune response. Our data show that some antimicrobial treatments can contribute to the evolution of phage-resistant pathogens with high virulence.

microbiology↗

bioRxiv2021

CRISPR-Cas is associated with fewer antibiotic resistance genes in bacterial pathogens

The acquisition of antibiotic resistance genes via horizontal gene transfer is a key driver of the rise in multidrug resistance amongst bacterial pathogens. Bacterial defence systems per definition restrict the influx of foreign genetic material, and may therefore limit the acquisition of antibiotic resistance. CRISPR-Cas adaptive immune systems are one of the most prevalent defences in bacteria, found in roughly half of bacterial genomes, but it has remained unclear if and how much they contribute to restricting the spread of antibiotic resistance. We analysed ~40,000 whole genomes comprising the full RefSeq dataset for 11 species of clinically important genera of human pathogens including Enterococcus, Staphylococcus, Acinetobacter and Pseudomonas. We modelled the association between CRISPR-Cas and indicators of horizontal gene transfer, and found that pathogens with a CRISPR-Cas system were less likely to carry antibiotic resistance genes than those lacking this defence system. Analysis of the mobile genetic elements targeted by CRISPR-Cas supports a model where this host defence system blocks important vectors of antibiotic resistance. These results suggest a potential "immunocompromised" state for multidrug-resistant strains that may be exploited in tailored interventions that rely on mobile genetic elements, such as phage or phagemids, to treat infections caused by bacterial pathogens.

microbiology↗

bioRxiv2020

Immune Lag Is a Major Cost of Prokaryotic Adaptive Immunity During Viral Outbreaks

CRISPR-Cas adaptive immune systems enable bacteria and archaea to efficiently respond to viral pathogens by creating a genomic record of previous encounters. These systems are broadly distributed across prokaryotic taxa, yet are surprisingly absent in a majority of organisms, suggesting that the benefits of adaptive immunity frequently do not outweigh the costs. Here, combining experiments and models, we show that a delayed immune response which allows viruses to transiently redirect cellular resources to reproduction, which we call "immune lag", is extremely costly during viral outbreaks, even to completely immune hosts. Critically, the costs of lag are only revealed by examining the early, transient dynamics of a host-virus system occurring immediately after viral challenge. Lag is a basic parameter of microbial defense, relevant to all intracellular, post-infection antiviral defense systems, that has to-date been largely ignored by theoretical and experimental treatments of host-phage systems.

microbiology↗

bioRxiv2019

Host resistance diversity protects susceptible genotypes by restricting pathogen spread and evolution

Diversity in host resistance often associates with reduced pathogen spread. This may result from ecological and evolutionary processes, likely with feedback between them. Theory and experiments on bacteria-phage interactions have shown that genetic diversity of the bacterial adaptive immune system can limit phage evolution to overcome resistance. Using the CRISPR-Cas bacterial immune system and lytic phage, we engineered a host-pathogen system where each bacterial host genotype could be infected by only one phage genotype. With this model system, we explored how CRISPR diversity impacts the spread of phage when they can overcome a resistance allele, how immune diversity affects the evolution of the phage to increase its host range, and if there was feedback between these processes. We show that increasing CRISPR diversity benefits susceptible bacteria via a dilution effect, which limits the spread of the phage. We suggest that this ecological effect impacts the evolution of novel phage genotypes, which then feeds back into phage population dynamics.

evolutionary biology↗