Biology subjects

Gurney, J.

Publications and source records attributed to Gurney, J..

11 recordsLinked to original sources

bioRxiv2026

The Phage Time Continuum: Genome Organization Constrains Anti-CRISPR Success

Bacteria and lytic phages experience antagonistic co-evolution, with bacterial defenses (e.g., CRISPR-Cas) battling phage anti-defenses (e.g., anti-CRISPR (Acr) systems). We investigate whether CRISPR-Cas and Acr engage in a timing-dependent genetic conflict. If anti-CRISPR genes act too late in infection, they risk failing to neutralize CRISPR, if Cas cannot find its target in time, Acr may be able to neutralize it. Because phages typically inject their genomes in a linear manner, location along the genome acts as a proxy for time from start of infection. Given these restrictions, we suggest that phage genomes that contain Acr systems will have a distinct pattern of spacers when compared to those that do not, reflecting the time dependent interaction of infections, we have termed this the Phage Time Continuum. In our analysis of 1462 phage genomes and 105 mobile plasmid genomes infecting Pseudomonas aeruginosa, we found clear distribution of where P. aeruginosa spacers map back onto these mobile genetic elements genomes. Further CRISPR spacers significantly enriched against anti-CRISPR systems over other anti-defense systems from both phage and plasmids. Finally, in genomes with anti-CRISPR, spacers map to earlier positions, suggesting that success hinges not just on targeting a given gene, but where those spacers hit along a genome.

microbiology↗

bioRxiv2026

Robust Inference of Phage Host Infection Dynamics from Sparse Single-Cell Transcriptomic Profiles in Pseudomonas aeruginosa

Phage therapy is providing a benefit to a limited number of people. To date those who have received phage therapy typically have done so via an IND expanded access mechanism, colloquially referred to as compassionate release, new clinical trials are underway, and the advent of phage therapy seems to be at hand. However, a recurring question remains, which phages to use for which bacteria. By understanding how a phage interacts with its bacterial host we will move closer to being able to provide an answer to this pressing question. By using bacterial single cell RNA sequencing (scRNAseq) during phage infection we can look at the most fundamental interaction between the phage and its host. We have observed phage gene expression consistent with well described expression profiles seen in bulk expression data. Phage genes in our scRNAseq data followed the early, mid, and late gene expression profile. Using a machine learning algorithm (Support Vector Machine) we were able to distinguish time of infection from phage genes. We also observed that phage infection is likely not random. When infected with two genetically distinct phages the likelihood of finding both inside a single cell was not consistent with Poisson distribution.

microbiology↗

bioRxiv2026

Sociality changes Gene Essentiality

Transposon insertion sequencing (Tn-seq) has become a powerful tool for assigning gene fitness and essentiality in bacteria, and has recently been extended to bacteriophages. A core but rarely examined assumption of these screens is that fitness is measured in an asocial environment, where each mutant succeeds or fails on its own. Yet many genes act socially: their products can be shared among neighbors, allowing defective mutants to be complemented in trans. In phages multiple genotypes routinely coinfect the same cell. Here we show that social interactions distort gene essentiality. Using paired quorum-sensing microarray and Tn-seq data from Pseudomonas aeruginosa, we find that quorum-sensing-regulated genes are over-represented among genes scored as non-essential, confirming that social genes are under-reported as essential. We then build a stochastic, agent-based model of phage Tn-seq across an MOI gradient, assigning each gene an intrinsic fitness effect and a complementation fraction. Complementable ("social") genes rise in frequency as MOI increases, masking their true fitness cost, whereas non-complementable ("private") genes, do not. Partitioning genes by life-cycle stage and applying a two-round high-then-low-MOI design, further separates gene functions by life cycle stage. We argue that deliberate MOI manipulation turns a confound into a tool, enabling systematic classification of phage sociality.

evolutionary biology↗

bioRxiv2024

Phage Steering in the Presence of a Competing Bacterial Pathogen

The rise of antibiotic-resistant bacteria has necessitated the development of alternative therapeutic strategies such as bacteriophage therapy, where viruses infect bacteria, reducing bacterial burden. However, rapid bacterial resistance to phage treatment remains a critical challenge, potentially leading to failure. Phage Steering, which leverages the evolutionary dynamics between phage and bacteria, offers a novel solution by driving bacteria to evolve away from virulence factors or resistance mechanisms. In this study, we examined whether Phage Steering using bacteriophage Luz19 could function in the presence of a competing pathogen, Staphylococcus aureus (USA300), while targeting Pseudomonas aeruginosa (PAO1). Through in vitro co-evolution experiments with and without the competitor, we observed that Luz19 consistently steered P. aeruginosa away from the type IV pilus (T4P), a key virulence factor, without interference from S. aureus. Genomic analyses revealed mutations in T4P-associated genes, including pilR and pilZ, which conferred phage resistance. Our findings suggest that Phage Steering remains effective even in polymicrobial environments, providing a promising avenue for enhancing bacteriophage therapy efficacy in complex infections. ImportancePhage Steering--using phages that bind essential virulence or resistance-associated structures-- offers a promising solution by selecting for resistance mutations that attenuate pathogenic traits. However, it remains unclear whether this strategy remains effective in polymicrobial contexts, where interspecies interactions may alter selective pressures. Here, we demonstrate that Pseudomonas aeruginosa evolves phage resistance via loss-of-function mutations in type IV pilus (T4P) when challenged with the T4P-binding phage Luz19, and that this evolutionary trajectory is preserved even in the presence of a competing pathogen, Staphylococcus aureus. Phage resistance was phenotypically confirmed via twitching motility assays and genotypically via whole genome sequencing. These findings support the robustness of Phage Steering under interspecies competition, underscoring its translational potential for managing complex infections--such as those seen in cystic fibrosis--where microbial diversity is the norm.

microbiology↗

bioRxiv2024

Community-wide interactions sustain life in geothermal spring habitats

We investigated an alga-dominated geothermal spring community in Yellowstone National Park, USA. Our goal was to determine how cells cope with abiotic stressors during diurnal sampling that spanned over two orders of magnitude in solar irradiance. We report a community level response to toxic metal resistance and energy cycling that spans the three domains of life. Arsenic detoxification is accomplished via complementary gene expression by different lineages. Photosynthesis is dominated by Cyanidioschyzon, with the mixotroph, Galdieria, relegated to nighttime heterotrophy. Many key functions, including the cell cycle, are strongly regulated by diurnal light fluctuations. These results demonstrate that biotic interactions are highly structured in extreme habitats. We suggest this was also the case on the early Earth when geothermal springs were cradles of microbial life, prior to the origin of eukaryotes.

evolutionary biology↗

bioRxiv2023

The las and rhl Quorum Sensing Systems in Pseudomonas aeruginosa Form a Multi-Signal Reciprocal Network Which Can Tune Reactivity to Variations in Physical and Social Environments

Researchers often view the multi-signal quorum sensing systems of Pseudomonas aeruginosa as a hierarchy, topped by the las system which acts as a master regulator. By experimentally controlling the concentration of auto-inducer signals in a signal null strain (PAO1{Delta}lasI{Delta}rhlI), we show that the two primary quorum sensing systems--las and rhl--act reciprocally rather than hierarchically. Just as the las systems 3-oxo-C12-HSL can induce increased expression of rhlI, the rhl systems C4-HSL increases the expression level of lasI. We develop a mathematical model to quantify relationships both within and between the las and rhl quorum sensing systems and the downstream genes they influence. The results show that not only do the systems interact reciprocally, but they do so cooperatively and nonlinearly, with the combination of C4-HSL and 3-oxo-C12-HSL increasing expression level far more than the sum of their individual effects. We computationally assess how our parameterized model responds to variation in social (population density) and physical (mass transfer) environment and demonstrate that a reciprocal architecture is more responsive to density and more robust to mass transfer than a strict hierarchy.

microbiology↗

bioRxiv2022

The dynamic response of quorum-sensing to density is robust to signal supplementation and signal synthase knockouts

Quorum sensing (QS) is a widespread mechanism of environment sensing and behavioral coordination in bacteria. At its core, QS is based on the production, sensing and response to small signaling molecules. Previous work with Pseudomonas aeruginosa shows that QS can be used to achieve quantitative resolution and deliver a dosed response to the bacterias density environment, implying a sophisticated mechanism of control. To shed light on how the mechanistic signal components contribute to graded responses to density, we assess the impact of genetic (AHL signal synthase deletion) and/or signal supplementation (exogenous AHL and PQS addition) perturbations on lasB reaction-norms to changes in density. Our approach condenses data from 2,000 timeseries (over 74,000 individual observations) into a comprehensive view of QS-controlled gene expression across variation in genetic, environmental, and signal determinants of lasB expression. We first confirm that deleting either ({Delta}lasI, {Delta}rhlI) or both ({Delta}lasIrhlI) signal synthase genes attenuates QS response. In the {Delta}rhlI background we show persistent yet attenuated density-dependent lasB expression due to native 3-oxo-C12 signaling. We then test if density-independent quantities of signal (3-oxo-C12, C4, PQS or combined) added to the WT either flatten or increase the reaction norm and find that the WT response is robust to all tested concentrations of signal, alone or in combination. We then move to progressively supplementing the genetic knockouts and find that cognate signal supplementation ({Delta}lasI+3-oxo-C12, {Delta}rhlI+C4) is sufficient to restore lasB expression and as well as reactivity to density encoded by the intact signal synthase. We also find that dual supplementation of the double synthase knockout restores expression but does not flatten the reaction norm. Despite adding a density-independent amount of AHL, the double signal synthase can still quantitively sense density. Our results show that a positive reaction norm to density is robust to multiple combinations of gene deletion and density-independent signal supplementation and that while density-independent signal supplementation can increase mean expression, the WT QS still retains the ability to quantitatively respond to density. Our work develops a modular approach to query the robustness and mechanistic bases of the central environmental sensing phenotype of quorum sensing.

microbiology↗

bioRxiv2021

Building reusable phage and antibiotic treatments via exploitation of bacteria-phage coevolutionary dynamics.

People with chronic (long-lasting) infections face the problem that treatment options diminish in time as the pathogen evolves increasing resistance. To address this challenge, we exploit phage and bacterial co-evolution, producing dynamic selection pressures that can return the pathogen to a state of susceptibility to the initial (regulator-approved) therapy. We show that phage OMKO1 alone triggers Arms Race Dynamic (ARD) co-evolution with the pathogen Pseudomonas aeruginosa, leading to generalized phage resistance and crucially - failure at reuse. In contrast, co-administration of the phage with antibiotics triggers Fluctuating Selection Dynamics (FSD) co-evolution, allowing for effective reuse after 20 days of treatment. We pursue medical relevance in our experiments with the use of clinically important pathogens, antibiotics, phage, and a benchmarked synthetic sputum medium. Phenotypic and genomic characterization of evolved isolates demonstrates that efflux-targeting phage OMKO1 exerts continued selection for antibiotic susceptibility regardless of co-evolutionary dynamic or antibiotic co-treatment, opening the door for evolutionary robust phage therapy.

microbiology↗

bioRxiv2019

Phage steering of antibiotic-resistance evolution in the bacterial pathogen Pseudomonas aeruginosa

Antimicrobial resistance is a growing global concern and has spurred increasing efforts to find alternative therapeutics. Bacteriophage therapy has seen near constant use in eastern Europe since its discovery over a century ago. One promising approach is to use phages that not only reduce bacterial pathogen loads, but also select for phage resistance mechanisms that trade-off with antibiotic resistance - so called phage steering. Recent work has shown that phage OMKO1 can interact with efflux pumps and in so doing select for both phage resistance and antibiotic sensitivity. We tested the robustness of this approach to three different antibiotics in vitro and one in vivo. We show that in vitro OMKO1 can reduce antibiotic resistance either in the absence or the presence of antibiotics. Our in vivo experiment showed that phage increased the survival times of wax moth larvae and increased bacterial sensitivity to erythromycin, both in the absence and presence of the antibiotic. We discuss the implications of our findings for future research on this promising therapeutic approach using OMKO1.

evolutionary biology↗

bioRxiv2019

In silico bacteria evolve robust cooperation via complex quorum-sensing strategies

Many species of bacteria collectively sense and respond to their social and physical environment via quorum sensing (QS), a communication system controlling extracellular cooperative traits. Despite detailed understanding of the mechanisms of signal production and response, there remains considerable debate over the functional role(s) of QS: in short, what is it for? Experimental studies have found support for diverse functional roles: density sensing, mass-transfer sensing, genotype sensing, etc. While consistent with theory, these results cannot separate whether these functions were drivers of QS adaption, or simply artifacts or spandrels of systems shaped by distinct ecological pressures. The challenge of separating spandrels from drivers of adaptation is particularly hard to address using extant bacterial species with poorly understood current ecologies (let alone their ecological histories). To understand the relationship between environmental challenges and trajectories of QS evolution, we used an agent-based simulation modeling approach. Given genetic mixing, our simulations produce behaviors that recapitulate features of diverse microbial QS systems, including coercive (high signal / low response) and generalized reciprocity (signal auto-regulation) strategists -- that separately and in combination contribute to QS-dependent resilience of QS-controlled cooperation in the face of diverse cheats. We contrast our in silico results with bacterial QS architectures that have evolved under largely unknown ecological contexts, highlighting the critical role of genetic constraints in shaping the shorter term (experimental evolution) dynamics of QS. More broadly, we see experimental evolution of digital organisms as a complementary tool in the search to understand the emergence of complex QS architectures and functions.\n\nAuthor summaryBacteria communicate and cooperate using complex cell-cell signaling systems known as quorum-sensing (QS). While the molecular mechanisms are often well understood, the reasons why bacteria use QS are less clear -- how has QS aided survival and growth? The answer to this question is dependent on the environment of adaptation, and unfortunately our current understanding of QS bacterial ecology is broadly lacking. To address this gap, we studied the evolution of digital organisms, individual-based computer simulations of bacterial populations evolving under defined environmental contexts. Our results pinpoint how simple environmental challenges (variable density and genetic mixing) can lead to the emergence of complex strategies that recapitulate features of bacterial QS, and open a path towards reverse-engineering the environmental drivers of QS.

microbiology↗

bioRxiv2018

Combinatorial quorum sensing in Pseudomonas aeruginosa allows for novel cheating strategies

In the opportunistic pathogen Pseudomonas aeruginosa, quorum sensing (QS) is a social trait that is exploitable by non-cooperating cheats. Previously it has been shown that by linking QS to the production of both public and private goods, cheats can be prevented from invading populations of cooperators and this has been termed a metabolic incentive to cooperate. We hypothesized P. aeruginosa could evolve novel cheating strategies to circumvent private goods metabolism by rewiring its combinatorial response to two QS signals (3O-C12-HSL and C4-HSL). We performed a selection experiment that cycled P. aeruginosa between public and private goods growth media and evolved an isolate which rewired its control of cooperative protease expression from a synergistic (AND-gate) response to dual signal input, to a 3O-C12-HSL only response. We show that this isolate circumvents metabolic incentives to cooperate and acts as a combinatorial signaling cheat, with a higher fitness in competition with its ancestor. Our results show three important principles; first, combinatorial QS allows for diverse social strategies to emerge; second, that restrictions levied by private goods are not sufficient to explain the maintenance of cooperation in natural populations and third that modifying combinatorial QS responses could result in important physiological outcomes in bacterial populations.

microbiology↗