Biology subjects

Brockhurst, M. A.

Publications and source records attributed to Brockhurst, M. A..

14 recordsLinked to original sources

bioRxiv2026

Adaptation to free-living drives loss of beneficial endosymbiosis through metabolic trade-offs

Symbioses are widespread (1) and underpin the function of diverse ecosystems (2-6), but their evolutionary stability is challenging to explain (7,8). Fitness trade-offs between contrasting intracellular and extracellular niches could act to stabilise endosymbioses because adaptation to either niche is predicted to reduce fitness in the alternate niche, thus reinforcing symbiosis (8,9). Here, we experimentally evolved four diverse Chlorella green algal endosymbionts of Paramecium bursaria to free-living conditions supplying either an amino acid, as provisioned by hosts (10,11), or nitrate, as available in freshwater (12), as the sole nitrogen source. Experimental algal populations adapted to free-living environments, generally increasing in population density and cellular chlorophyll content over time. In one of the four endosymbiont strains, adaptation to the nitrate free-living environment, but not the amino acid environments, drove the loss of fitness benefits to the host in reconstituted symbioses. This loss was not associated with reduced ability to grow on host-provisioned amino acids, nor lost ability to release the sugars provisioned to the host (10,13). Genome sequencing of evolved algal lines revealed genomic divergence between nitrate-adapted and amino acid-adapted lines, affecting genes involved with metabolic organisation and intracellular resource transport. Untargeted metabolomic profiling further showed extensive changes to membrane remodelling and turnover in N-evolved lines. Together, our data support a role for metabolic trade-offs driving divergence between contrasting intracellular and extracellular niches, with nitrogen as a key environmental axis driving divergence. Fitness trade-offs may, therefore, be a general, simple mechanism acting to reinforce symbiosis, contributing to evolutionary stability.

evolutionary biology↗

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↗

bioRxiv2026

Using auxotrophic donor strains to explore pQBR57 plasmid host range among environmental soil bacterial isolates

Plasmid host range (PHR) plays a key role in the spread of ecologically important genes, alongside applications in microbiome engineering, and environmental biotechnology. PHR is a complex trait arising from the combination of plasmid, donor and recipient properties. Most studies of PHR use a single donor strain, leaving the role of the donor unexplored, and often require genetically tagged recipient strains for counter selection, which limits use of non-genetically tractable strains. Here we developed a PHR screening method using auxotrophic donors that bypasses the need to genetically tag recipients, thus allowing the screening of culturable environmental bacterial strains. Specifically, we used two auxotrophic donors (P. fluorescens and P. putida), and the plasmid pQBR57-tphKAB, an environmental plasmid engineered for terephthalic acid bioremediation. We screened a library of 101 soil isolates, as potential recipients, including common soil genera of soil bacteria, Pseudomonas, Bacillus and Xanthomonas. We only observed conjugation into other Pseudomonas, but donor identity affected PHR, with P. fluorescens conjugating the plasmid into more recipient strains than P. putida. Phylogenomic analysis revealed that transconjugants clustered with P. citronellosis and P. putida lineages. In strains that were close relatives of transconjugants but who were unable to acquire the plasmid, we observed 5 defence systems not present in transconjugants that may act as barriers to plasmid acquisition. Our method provides a rapid, tag-free framework for screening PHR in environmental isolates and for investigating the influence of donor identity on plasmid conjugation.

microbiology↗

bioRxiv2025

Trade-offs between phage resistance and conjugative ability shape the ecological and evolutionary response of a multidrug resistance plasmid to plasmid-dependent phage

Phage therapy is a promising alternative to antibiotics to treat multidrug resistant infections. Plasmid dependent phages (PDPs) are particularly attractive as therapeutics because they can both kill targeted pathogen cells, whilst also potentially preventing the further spread of antibiotic resistance genes encoded by plasmids. However, we lack experimental studies of the ecological and evolutionary response of multidrug resistance plasmids against plasmid dependent phage treatment under ecologically relevant scenarios allowing plasmid conjugation. We experimentally evolved populations of E. coli carrying the multidrug resistance RP4 plasmid with the PRD1 PDP under conditions where conjugation was associated with either strong or weak benefits. When opportunities for conjugation were rare, PRD1 only transiently suppressed the conjugative plasmid population due to the rapid evolution of PRD1 resistant plasmids that lacked conjugative ability. Increasing the ecological opportunity for conjugation enhanced plasmid suppression and delayed the evolution of PRD1 resistant plasmids. PRD1 resistance was associated with reduced conjugative ability, but this trade-off was complex due to the heterogeneous impacts of resistance mutations on pilus production and conjugative ability. Mutations and IS element insertions in conjugation genes caused a wide range of PRD1 resistance phenotypes, ranging from complete resistance (virB4) to partial resistance (trbB, trbL). Bioinformatic analysis of publicly available IncP plasmid sequences showed that truncated variants of VirB4 protein are common in natural populations, suggesting that plasmid-dependent phages are an important selective pressure in microbial communities. Our results demonstrate an evolutionary trade-off between conjugative ability and phage resistance that cannot be easily circumvented by plasmids. Targeting multidrug resistance plasmids with PDPs is likely to drive loss of conjugation limiting the transfer of antibiotic resistance genes in bacterial communities.

evolutionary biology↗

bioRxiv2025

A novel two-component system controls vancomycin resistance in epidemic Clostridioides difficile

The glycopeptide antibiotic vancomycin is the frontline treatment for C. difficile infection in the UK. There have been only sporadic reports of resistance in the clinic but testing is rare so the true resistance landscape is unclear. We have previously shown that resistance can emerge rapidly in vitro via distinct but complementary pathways. Strain Bc2, characterised here, is an experimentally evolved derivative of C. difficile strain R20291 that displays a 16-fold increase in vancomycin MIC over its parent. Bc2 has point mutations in dacS, bclA3, CDR20291_0794, CDR20291_1871 and CDR20291_3124 (vnrS). By genetically engineering a wild-type vancomycin susceptible strain, we demonstrated that a combination of just two mutations, dacSc.798A>T and vnrSc.692G>T, both of which encode two-component system histidine kinases, was sufficient to recapitulate Bc2 resistance. We have previously shown that mutations in dacS can confer low level resistance via increases in the expression of a D,D-carboxypeptidase DacJ. dacSc.798A>T also led to increased transcription of dacJ and led to a modest increase in vancomycin MIC. Surprisingly vnrSc.692G>T led to overexpression of the vanG cluster, which encodes all of the enzymes needed for resistance via substitution of the terminal D-Ala on peptidoglycan lipid II precursors with D-Ser. Genomic analysis of a large collection of European C. difficile strains showed that a three gene cluster, which includes vnrS, vnrR (encoding the cognate response regulator) and an adjacent gene CDR20291_3123, is unique to the phylogenetic branch that contains strains belonging to epidemic ribotypes 027 and 176. Transcriptomic analysis of the wider VnrS regulon also revealed an additional previously unknown role in regulating the expression of the flagellum, an important virulence factor in C. difficile. Analysis of the response to vancomycin exposure also revealed that dacJ is one of a small set of genes that are upregulated shortly after antibiotic stress, even in the absence of mutations that typically lead to its overexpression. Together these data reveal a new synergistic route, needing only two point mutations, by which epidemic lineages of C. difficile can attain vancomycin resistance. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=147 HEIGHT=200 SRC="FIGDIR/small/671617v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@bba369org.highwire.dtl.DTLVardef@e562acorg.highwire.dtl.DTLVardef@1b112deorg.highwire.dtl.DTLVardef@4dcf8a_HPS_FORMAT_FIGEXP M_FIG C_FIG

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

Chromosomal resistance mutations facilitate acquisition of multidrug-resistance plasmids in Escherichia coli

Bacteria can gain multiple resistance mechanisms in a single step by the acquisition of multidrug-resistance (MDR) plasmids, but it is unclear how antibiotic selection during the acquisition of MDR plasmids affects the evolution of additional resistance mechanisms. Through conjugating separate ESBL- and carbapenamase-producing MDR plasmids into plasmid-naive Escherichia coli hosts we examine the effects of acquisition of a single plasmid or co-acquisition of multiple plasmids upon fitness costs, resistance and subsequent genomic adaptation. We show that acquisition of pOXA-48, encoding OXA-48 carbapenamase, is associated with highly variable fitness costs and levels of resistance to ertapenem in transconjugants independent of the presence of pLL35. This phenomenon was not observed during the acquisition of ESBL CTX-M-15 encoding pLL35 alone. Transconjugants receiving pOXA-48 rapidly gained parallel mutations affecting the membrane porin OmpF, or its regulators OmpR or EnvZ. These chromosomal mutations were not compensatory for the fitness costs imposed by the plasmid, nor did they provide significant increases in resistance to carbapenems in the absence of the pOXA-48. Rather, they acted synergistically with the plasmid-encoded carbapenamase, which alone only provided marginal resistance, together providing high-level resistance to ertapenem. Such rapid evolutionary processes may play an important role in plasmid dynamics within environments with strong antibiotic selection for plasmid-encoded ARGs, particularly when these ARGs provide only marginal resistance.

microbiology↗

bioRxiv2024

Immune-like glycan-sensing and horizontally-acquired glycan-processing orchestrate host control in a microbial endosymbiosis

Endosymbiosis was a key factor in the evolution of eukaryotic cellular complexity. Yet the mechanisms that allow host regulation of intracellular symbionts, a pre-requisite for stable endosymbiosis and subsequent organelle evolution, are largely unknown. Here, we describe an immune-like glycan-sensing/processing network, partly assembled through horizontal gene-transfers (HGTs), that enables Paramecium bursaria to control its algal endosymbionts. Using phylogenetics, RNA-interference (RNAi), and metabolite exposure experiments, we show that P. bursaria regulates endosymbiont destruction using glycan-sensing/processing - a system that includes a eukaryotic-wide chitin-binding chitinase-like protein (CLP) localized to the host phago-lysosome. RNAi of CLP alters expression of eight glycan-processing genes, including two prokaryote-derived HGTs, during endosymbiont destruction. Furthermore, glycan-sensing/processing dynamically regulates endosymbiont number in P. bursaria, plasticity crucial to maximize host fitness across ecological conditions. CLP is homologous to a human phagocyte-associated innate immune factor, revealing how immune functions can be alternatively adapted and expanded, partly through HGT, enabling endosymbiotic control. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=186 HEIGHT=200 SRC="FIGDIR/small/613017v2_ufig1.gif" ALT="Figure 1"> View larger version (77K): org.highwire.dtl.DTLVardef@13aeaaorg.highwire.dtl.DTLVardef@302e6dorg.highwire.dtl.DTLVardef@138224forg.highwire.dtl.DTLVardef@54aca8_HPS_FORMAT_FIGEXP M_FIG C_FIG

evolutionary biology↗

bioRxiv2024

Global genomic diversity of Pseudomonas aeruginosa in bronchiectasis

BackgroundPseudomonas aeruginosa is the dominant pathogen causing lung infections in people with both cystic fibrosis (CF) and bronchiectasis, associated with poorer outcomes. Unlike CF, bronchiectasis has been a neglected disease. More extensive genomic studies of larger bronchiectasis patient cohorts and within patient sampling are needed to improve understanding of the evolutionary mechanisms underpinning P. aeruginosa infections to guide novel and improved treatments. MethodsWe have performed genome sequencing of 2,854 P. aeruginosa isolates from 180 patients attending clinics worldwide to analyse the genomic diversity between and within patient infections. ResultsWe observed high genetic diversity between infections with low incidence of highly transmissible strains. Our genomic data provide evidence for the mutational targets driving P. aeruginosa evolution in bronchiectasis. Some functions found to gain mutations were comparable to CF, including biofilm and iron acquisition, whilst others highlighted distinct evolutionary paths in bronchiectasis such as pyocin production and resistance, and a novel efflux pump gene (PA1874). We also show a high incidence of antimicrobial resistance-associated mutations and acquired resistance genes, in particular multidrug efflux and fluoroquinolone resistance mechanisms. ConclusionsOur findings highlight important differences between P. aeruginosa infections in bronchiectasis and CF and provide evidence of the relatively minor role transmissible strains play in bronchiectasis. Our study provides a 10-fold increase in the available genomic data for these infections and is a global resource to improve our knowledge and understanding, to facilitate better patient outcomes. SummaryThe largest genomic study of Pseudomonas aeruginosa bronchiectasis isolates to-date, providing an unprecedented global genomic resource. We highlight important differences between bronchiectasis and cystic fibrosis, including key genes under selection.

genomics↗

bioRxiv2024

Superiority of chromosomal compared to plasmid-encoded compensatory mutations

Plasmids are important vectors of horizontal gene transfer in microbial communities but can impose a burden on the bacteria that carry them. Such plasmid fitness costs are thought to arise principally from conflicts between chromosomal- and plasmid-encoded molecular machineries, and thus can be ameliorated by compensatory mutations (CMs) that reduce or resolve the underlying causes. CMs can arise on plasmids (i.e. plaCM) or on chromosomes (i.e. chrCM), with contrasting predicted effects upon plasmid success and subsequent gene transfer because plaCM can also reduce fitness costs in plasmid recipients, whereas chrCM can potentially ameliorate multiple distinct plasmids. Here, we develop theory and a novel experimental system to directly compare the ecological effects of plaCM and chrCM that arose during evolution experiments between Pseudomonas fluorescens SBW25 and its sympatric mercury resistance megaplasmid pQBR57. We show that while plaCM was predicted to succeed under a broader range of parameters in mathematical models, experimentally chrCM dominated under all conditions, including those with numerous recipients, due to a more efficacious mechanism of compensation, and advantages arising from transmission of costly plasmids to competitors (plasmid weaponisation). We show analytically the presence of a mixed Rock-Paper-Scissors regime for plaCM, driven by trade-offs with horizontal transmission, that explains the observed failure of plaCM to dominate even in competition against an uncompensated plasmid. Our results reveal broader implications of plasmid-bacterial evolution for plasmid ecology, demonstrating the importance of compensatory mutations for resistance gene spread. One consequence of the superiority of chrCM over plaCM is the likely emergence in microbial communities of compensated bacteria that can act as hubs for plasmid accumulation and dissemination.

microbiology↗

bioRxiv2023

Multiple evolutionary pathways lead to vancomycin resistance in Clostridioides difficile

Clostridioides difficile is an important human pathogen, for which there are very limited treatment options, primarily the glycopeptide antibiotic vancomycin. In recent years vancomycin resistance has emerged as a serious problem in several Gram positive pathogens, but high level resistance has yet to be reported for C. difficile, although it is not known if this is due to constraints upon resistance evolution in this species. Here we show that resistance to vancomycin can evolve rapidly under ramping selection but is accompanied by severe fitness costs and pleiotropic trade-offs, including sporulation defects that would be expected to severely impact transmission. We identified two distinct pathways to resistance, both of which are predicted to result in changes to the muropeptide terminal D-Ala-D-Ala that is the primary target of vancomycin. One of these pathways involves a previously uncharacterised D,D-carboxypeptidase, expression of which is controlled by a dedicated two-component signal transduction system. Our findings suggest that while C. difficile is capable of evolving high-level vancomycin resistance, this outcome may be limited clinically due to pleiotropic effects on key pathogenicity trains. Moreover, our data provide a mutational roadmap to inform genomic surveillance.

microbiology↗

bioRxiv2023

Genome dynamics across the evolutionary transition to endosymbiosis

Endosymbiosis - where a microbe lives and replicates within a host - is an important contributor to organismal function that has accelerated evolutionary innovations and catalysed the evolution of complex life. The evolutionary processes associated with transitions to endosymbiosis, however, are poorly understood. Here, we use comparative genomics of the genus Arsenophonus to reveal the complex processes that occur on evolution of an endosymbiotic lifestyle. We compared the genomes of 38 strains spanning diverse lifestyles from environmentally acquired infections to obligate inter-dependent endosymbionts. We observed recent endosymbionts had larger genome sizes than closely related environmentally acquired strains, consistent with evolutionary innovation and rapid gain of new function. Increased genome size was a consequence of prophage and plasmid acquisition including a cargo of type III effectors, and concomitant loss of CRISPR-Cas genome defence systems enabling mobile genetic element expansion. Persistent endosymbiosis was also associated with loss of type VI secretion, likely reflecting reduced microbe-microbe competition. Thereafter, the transition to stable endosymbiosis and vertical inheritance was associated with the expected relaxation of purifying selection, pseudogenisation of genes and reduction of metabolism, leading to genome reduction. However, reduced %GC that is typically considered a progressive linear process was observed only in obligate interdependent endosymbionts. We argue that a combination of the need for rapid horizontal gene transfer-mediated evolutionary innovation together with reduced phage predation in endosymbiotic niches drives loss of genome defence systems and rapid genome expansion upon adoption of endosymbiosis. These remodelling processes precede the reductive evolution traditionally associated with adaptation to endosymbiosis.

genomics↗

bioRxiv2022

Horizontal gene transfer increases microbiome stability

Genes encoding resistance to stressors, such as antibiotics or environmental pollutants, are widespread across microbiomes, often encoded on mobile genetic elements. Yet despite their prevalence, the impact of resistance genes and their mobility upon the dynamics of microbial communities remains largely unknown. Here we develop eco-evolutionary theory to explore how resistance genes alter the stability of diverse microbiomes. We show that adding resistance genes to a microbiome typically increases its overall stability, particularly for mobile resistance genes with high transfer rates that efficiently spread resistance throughout the community. However, the impact of resistance genes upon the stability of individual taxa varies depending upon the mobility of the resistance gene and the network of ecological interactions within the community. Non-mobile resistance genes can benefit susceptible taxa in cooperative communities, yet damage those in competitive communities. Moreover, whilst the transfer of mobile resistance genes generally increases the stability of previously susceptible recipient taxa to perturbation, it can, counterintuitively, decrease the stability of the originally resistant donor species. We confirmed these theoretical predictions experimentally using competitive soil microcosm communities. Here the stability of a susceptible microbial community to perturbation was increased by adding mobile resistance genes encoded on conjugative plasmids but was decreased when these same genes were encoded on the chromosome. Together these findings highlight the importance of horizontal gene transfer in driving the eco-evolutionary dynamics of diverse microbiomes.

evolutionary biology↗

bioRxiv2019

Transposable temperate phages promote the evolution of divergent social strategies in Pseudomonas aeruginosa populations

Transposable temperate phages randomly insert into bacterial genomes, providing increased supply and altered spectra of mutations available to selection, thus opening alternative evolutionary trajectories. Transposable phages accelerate bacterial adaptation to new environments, but their effect on adaptation to the social environment is unclear. Here we show, using experimental evolution of Pseudomonas aeruginosa in iron-limited and iron-rich environments causing differential expression of siderophore cooperation, that transposable phages promoted divergence into extreme siderophore production phenotypes in iron-limited populations. Iron-limited populations with transposable phages evolved siderophore over-producing clones alongside siderophore non-producing cheats. Low siderophore production was associated with parallel mutations in pvd genes, encoding pyoverdine biosynthesis, and pqs genes, encoding quinolone signaling, while high siderophore production was associated with parallel mutations in phenazine-associated gene clusters. Notably, some of these parallel mutations were caused by phage insertional inactivation. These data suggest that transposable phages, which are widespread in microbial communities, can mediate the evolutionary divergence of social strategies.

evolutionary biology↗