Biology subjects

Rainey, P. B.

Publications and source records attributed to Rainey, P. B..

At least 19 recordsLinked to original sources

bioRxiv2026

Forging an evolutionary individual from separate replicators

Major evolutionary transitions occur when independently replicating particles unite into a higher-level individual. How selection comes to work on particle collectives and generates reliable heredity is unresolved. By packaging red and green plasmids in yeast cells we made plasmid communities units of selection: cell boundaries supplied discreteness, segregation generated variation, and cell division reproduced communities. Communities colouring cells yellow were preferentially propagated. Initially, yellow parents rarely produced yellow offspring, but continued selection aligned replicative fates of the two plasmids favouring recombinants that fused with the endogenous 2-micron element. Chimera stability was secured by mutations in FLP1, which encodes a recombinase that otherwise resolves fused replicons. Reliable collective heredity, here, a derived product of higher-level selection, marked completion of an evolutionary transition in individuality.

evolutionary biology↗

bioRxiv2026

Transcriptomic dissection of genetically diverse Wrinkly Spreader mutants reveals hidden layers of complexity

Understanding the genetic basis of adaptive traits requires linking mutation to phenotypic change and fit-ness. The Wrinkly Spreader (WS) mutants of Pseudomonas fluorescens SBW25, which colonize the air-liquid interface (ALI) in static microcosms, provide a model for dissecting genotype-phenotype relationships. Muta-tions generating the WS phenotype cause constitutive activation of regulatory pathways synthesising cyclic bis-(3,5)-dimeric guanosine monophosphate (c-di-GMP) leading to over-production of cellulose encoded by the wss operon. Using RNA-seq on 14 independently evolved WS mutants, each carrying a single characterised mutation in one of 14 different genes affecting production of c-di-GMP, we report that transcriptional responses are highly genotype specific, with few differentially expressed genes (DEGs) shared among WS mutants. Genes showing greatest variance in expression included fimbrial and matrix-associated loci such as fap, cupE and pga. fap and cupE were the only fimbriae-related loci active in these mutants. Targeted deletions of these candidate loci revealed subtle phenotypic and fitness effects. Closer examination of the fap locus showed that its expression is genotype dependent, restricted to a minority of cells, and associated with stability in early stages of mat formation. Overall, our results confirm that previous suppressor-based approaches have captured the major genetic components of the WS phenotype: cellulose encoded by the wss operon and regulated by elevated c-di-GMP. At the same time, it is apparent that morphology-based screens have failed to detect genes having more subtle effects, whose contributions influence success of WS mutants at the ALI. These findings reveal additional layers of complexity that underscore the polygenic nature of the WS phenotype.

microbiology↗

bioRxiv2025

Diverse microbial communities assemble on both recalcitrant and labile carbon sources

Microbial community assembly is shaped by the nature of available resources, with labile carbon sources such as glucose often expected to support low diversity due to rapid growth and competitive exclusion. In contrast, recalcitrant substrates like cellulose are thought to support higher diversity through slower growth and increased niche partitioning. In previous work, we showed that compost-derived microbial communities propagated on cellulose maintained high diversity over nearly a year. To determine whether such diversity is specific to recalcitrant substrates or reflects more general features of assembly, we tracked community dynamics in three environments--cellulose paper, cellulose broth, and glucose--using daily 16S rRNA profiling over eight weeks. Communities were maintained through bi-weekly serial transfers, with five replicates per treatment, yielding a high-resolution dataset of over 800 samples. Despite originating from the same inoculum, communities diverged sharply in both taxonomic and functional composition. Cellulose environments yielded stable communities enriched in specialists, while glucose environments exhibited rapid succession and dominance by generalists. Surprisingly, all environments sustained comparably high levels of taxonomic diversity. Functional inference suggested extensive cross-feeding and resource salvaging in both cases. While the mechanisms maintaining diversity on glucose remain unresolved, our results reveal distinct assembly trajectories under simple carbon regimes and provide a foundation for future mechanistic study.

ecology↗

bioRxiv2025

Context-Dependent Adaptation in Structured Environments

Adaptive evolution often leads to niche specialization, but successful colonization of a new niche can depend as much on ecological context as on genetic change. This is especially true in spatially structured environments, where the physical construction of habitat and the order of arrival of mutants can shape evolutionary outcomes. In static broth cultures, the air-liquid interface (ALI) provides a high-oxygen niche typically colonized by mat-forming mutants. In Pseudomonas fluorescens, a range of single mutations that upregulate cellulose production give rise to "wrinkly spreader" (WS) types--canonical niche specialists that out-compete ancestral types at the ALI. Yet we discover that this is a half-truth: WS mutants frequently fail to establish when introduced alone at low density, but co-culture with ancestral types rescues colonization. Microscopy and model simulations show that ancestors physically scaffold the ALI, enabling stable attachment and facilitating the in situ emergence of an array of genetically diverse WS mutants. Ancestors later disperse, vacate the niche allowing WS mutant expansion. This transient asymmetric interaction reveals that even strongly adaptive mutations may require ecological facilitation to succeed. More broadly, our findings challenge simple narratives of adaptive replacement and underscore how transient ecological dependencies can shape the trajectory of evolutionary change.

evolutionary biology↗

bioRxiv2024

Experimental capture of genomic islands defines a widespread class of genetic element capable of non-autonomous transfer

Bacteria acquire new genes by horizontal gene transfer (HGT), typically mediated by mobile genetic elements (MGEs). While plasmids, bacteriophages and certain integrative and conjugative elements (ICEs) are well characterized, the broader diversity of MGEs remains poorly understood. Here, we propagated the bacterium Pseudomonas fluorescens SBW25 in the presence of filtrate obtained from garden compost communities. Genome sequencing of derived colonies revealed acquisition of three different mobile elements, each integrated immediately downstream of tmRNA, flanked by direct repeats, and encoding a tyrosine integrase (intY) and putative phage defense systems. Absent are genes with recognized roles in autonomous transfer. Interrogation of DNA sequence databases showed that similar elements are widespread in the genus Pseudomonas and beyond, with Vibrio Pathogenicity Island-1 (VPI-1) from V. cholerae as a notable example. Bioinformatic analyses demonstrate frequent horizontal transfer among diverse hosts. Detailed analysis of a single element, I55, showed that it is transferred between cells by a jumbo phage, and confers a fitness benefit via a type II restriction-modification system. Significance StatementThe impact of horizontal gene transfer on the evolution of bacteria outpaces that driven by spontaneous mutation, but knowledge of the range of mediators, the genes mobilized, and the mechanisms of movement have largely depended on inferences stemming from bioinformatics. Here we describe a real-time evolution experiment in which a single focal strain propagated with filtrate from a complex microbial community captured genetic elements carrying a diverse cargo of genes whose mobility was previously uncharacterized. The elements represent a widespread class of mobile DNA dependent only on a tyrosine integrase targeting a highly conserved genomic site. Genetic analysis of one element shows that it confers a significant fitness advantage via defense against bacteriophages, and hijacks a jumbo bacteriophage for intercellular transfer. Our findings reveal new insights into the hidden diversity and dynamics of MGEs in natural environments. ClassificationBiological Sciences / Microbiology

microbiology↗

bioRxiv2024

An extreme mutational hotspot in nlpD depends on transcriptional induction of rpoS

Mutation rate varies within and between genomes. Within genomes, tracts of nucleotides, including short sequence repeats and palindromes, can cause localised elevation of mutation rate. Additional mechanisms remain poorly understood. Here we report an instance of extreme mutational bias in Pseudomonas fluorescens SBW25 associated with a single base-pair change in nlpD. These mutants frequently evolve in static microcosms, and have a cell-chaining (CC) phenotype. Analysis of 153 replicate populations revealed 137 independent instances of a C565T loss-of-function mutation at codon 189 (CAG to TAG (Q189*)). Fitness measures of alternative nlpD mutants showed molecular parallelism to be unconnected to selective advantage. Recognising that transcription can be mutagenic, and that codon 189 overlaps with a predicted promoter (rpoSp) for the adjacent stationary phase sigma factor, rpoS, transcription across this promoter region was measured. This confirmed rpoSp is induced in stationary phase and that C565T mutation caused significant elevation of transcription. The latter provided opportunity to determine the C565T mutation rate using a reporter-gene fused to rpoSp. Fluctuation assays demonstrate the C565T mutation rate to be 5,700-fold higher than expected. In Pseudomonas, transcription of rpoS requires the positive activator PsrA, which we show also holds for SBW25. Fluctuation assays performed in a {Delta}psrA background showed a 60-fold reduction in mutation rate confirming that the elevated rate of mutation at C565T mutation rate is dependent on induction of transcription. This hotspot suggests a generalisable phenomenon where the induction of transcription causes elevated mutation rates within defining regions of promoters.

evolutionary biology↗

bioRxiv2024

Evolutionary dynamics of nascent multicellular lineages

The evolution of multicellular organisms involves the emergence of cellular collectives that eventually become units of selection in their own right. The process can be facilitated by ecological conditions that impose heritable variance in fitness on nascent collectives with long-term persistence depending on capacity of competing lineages to transition reliably between soma- and germ-like stages of proto-life cycles. Prior work with experimental bacterial populations showed rapid increases in collective-level fitness with capacity to switch between life cycle phases being a particular focus of selection. Here we report experiments in which the most successful lineage from the earlier study was further propagated for 10 life cycle generations under regimes that required different investment in the somalike phase. To explore the adaptive significance of switching, a control was included in which reliable transitioning between life cycle phases was abolished. The switch proved central to maintenance of fitness. Moreover, in a non-switch treatment, where solutions to producing a robust and enduring soma-phase were required, evolution of mutL-dependent switching emerged de novo. A newly developed computational pipeline (colgen) was used to display the moment-by-moment evolutionary dynamics of lineages providing rare visual evidence of the roles of chance, history and selection. Colgen, underpinned by a Bayesian model, was further used to propagate hundreds of mutations back through temporal genealogical series, predict lineages and time points corresponding to changes of likely adaptive significance, and in one instance, via a combination of targeted sequencing, genetics and analyses of fitness consequences, adaptive significance of a single mutation was demonstrated. Overall, our results shed light on the mechanisms by which collectives adapt to new selective challenges and demonstrate the value of genealogy-centered approaches for investigating the dynamics of lineage-level selection.

evolutionary biology↗

bioRxiv2024

Experimental evolution of evolvability

Capacity to generate adaptive variation can evolve by natural selection. However, the idea that mutation becomes biased toward specific adaptive outcomes is controversial. Here, using experimental bacterial populations, we report the evolution of enhanced evolvability via localised hyper-mutation. Key to realisation was a lineage-level birth-death dynamic, where lineage success depended upon capacity to mutate between two phenotypic states, each optima in a cycling environment. The evolved mechanism is analogous to "contingency loci" in pathogenic bacteria, whose origin was previously unclear. Subsequent evolution showed lineages with localised hyper-mutability were more likely to acquire additional adaptive mutations. Our results provide a detailed mechanistic account of the adaptive evolution of evolvability.

evolutionary biology↗

bioRxiv2023

Eco-evolutionary dynamics of massive, parallel bacteriophage outbreaks in compost communities

Bacteriophages are important drivers of microbial ecosystems, yet their dynamics in complex natural communities remain poorly understood compared to simplified laboratory systems. To address this gap, we analyzed viral dynamics across 20 compost-derived microbial communities propagated for one year in mesocosms. Communities clustered into two distinct types, each dominated by different cellulose-degraders and comprising hundreds of genera. In one type, we observed massive, parallel outbreaks of Theomophage, a previously undescribed bacteriophage, reaching 74% of metagenomic sequencing reads, the largest bacteriophage outbreak documented to date. Theomophage populations in isolated communities were composed of a single genotype that showed striking evolutionary stability throughout the experiment. In contrast, following experimental viral migration between mesocosms, all Theomophage populations showed rapid evolution via recombination between preexisting genotypes, replacement of ancestral lineages, and, upon successful migration to mesocosms of the alternate community type where Theomophage was initially absent, rapid acquisition of novel mutations that swept local populations and then spread to other mesocosms. Our study reveals the spatial and temporal scales at which bacteriophage microdiversity evolves in complex communities. It further shows that mixing of viral communities -- likely common in natural systems -- can rapidly accelerate bacteriophage evolution.

microbiology↗

bioRxiv2023

Rapid dissemination of host-metabolism-manipulating transposon-like entities via integrative and conjugative elements

Integrative and conjugative elements (ICEs) are self-transmissible mobile elements that transfer functional genetic units across broad phylogenetic distances. Accessory genes shuttled by ICEs can make significant contributions to bacterial fitness, yet ICEs that carry accessory genes encoding functions other than antimicrobial resistance remain poorly characterized. Recent observation of the rapid acquisition of ICEs in a pandemic lineage of Pseudomonas syringae pv. actinidae led to investigation of the structural and functional diversity of these elements among a diverse array of P. syringae. Fifty-three unique ICE types were identified across multiple phylogroups. These ICEs display distinct evolutionary histories compared to their bacterial hosts, are highly recombinogenic, exhibit a conserved structure and are punctuated by hotspots of accessory gene integration. Many carry a 16 kb transposon-like entity (Tn6212) that shows little polymorphism indicating recent dissemination. Deletion of Tn6212 did not alter pathogen growth in planta, but mutants displayed significant fitness defects when grown on TCA cycle intermediates. These were largely attributable to a single LysR regulator. RNA-seq analysis of a set of nested Tn6212 deletions confirmed a central role of LysR in enhanced expression of more than 300 genes and down-regulation of genes controlling expression of energetically demanding loci. Together the transcriptional data indicate a major role for Tn6212 in manipulation of bacterial metabolism with primary effects on RNA degradation, protein synthesis and potential diversion of ATP to growth.

microbiology↗

bioRxiv2023

Identifying and tracking mobile elements in evolving compost communities yields insights into the nanobiome

Microbial evolution is driven by rapid changes in gene content mediated by horizontal gene transfer (HGT). While mobile genetic elements (MGEs) are important drivers of gene flux, the nanobiome - the zoo of Darwinian replicators that depend on microbial hosts - remains poorly characterised. New experimental approaches and analyses are necessary to advance our understanding beyond simple pairwise MGE-host interactions. To detect horizontal transfer, a bioinformatic pipeline (xenoseq) was developed to cross-compare metagenomic samples, which was then applied to metagenomic data from evolving compost communities. These communities were routinely exposed to an "MGE cocktail" derived from allopatric communities. We show that this results in horizontal transfer of a multitude of previously undetected MGEs, including bacteriophages, phage-plasmids, megaplasmids, and even nanobacteria. Sequences that spread from one community to another are shown to disproportionally carry characteristics of phages and insertion-sequences, i.e., traits of canonically parasitic MGEs. We also show that one particularly prolific mobile element - a 313 kb plasmid - correlates substantially with rates of ammonia production, which under nitrogen limitation is likely beneficial. Taken together, our data show that new experimental strategies combined with bioinformatic analyses of metagenomic data stand to provide insight into the drivers of microbial community evolution.

microbiology↗

bioRxiv2022

Barcoding populations of Pseudomonas fluorescens SBW25

In recent years evolutionary biologists have developed increasing interest in the use of barcoding strategies to study eco-evolutionary dynamics of lineages within evolving populations and communities. Although barcoded populations can deliver unprecedented insight into evolutionary change, barcoding microbes presents specific technical challenges. Here, strategies are described for barcoding populations of the model bacterium Pseudomonas fluorescens SBW25, including the design and cloning of barcoded regions, preparation of libraries for amplicon sequencing, and quantification of resulting barcoded lineages. In so doing, we hope to aid the design and implementation of barcoding methodologies in a broad range of model and non-model organisms.

molecular biology↗

bioRxiv2022

The Effect of Bottleneck Size on Evolution in Nested Darwinian Populations

Previous work has shown how a minimal ecological structure consisting of patchily distributed resources and recurrent dispersal between patches can scaffold Darwinian properties onto collections of cells. When the timescale of dispersal is long compared with the time to consume resources, patches evolve such that their size increases, but at the expense of cells whose growth rate decreases within patches. This creates the conditions that initiate evolutionary transitions in individuality. A key assumption of this scaffolding is that a bottleneck is created during dispersal, so patches are founded by single cells. The bottleneck decreases competition within patches and hence creates a strong hereditary link at the level of patches. Here we construct a fully stochastic model of nested Darwinian populations and investigate how larger bottlenecks affect the evolutionary dynamics at both cell and collective levels. It is shown that, up to a point, larger bottlenecks simply slow the dynamics, but at some point, which depends on the parameters of the within-patch model, the direction of evolution toward the equilibrium is reversed. Introducing random bottleneck sizes with some positive probability of smaller sizes can counteract this, even if the probability of smaller bottlenecks is small.

evolutionary biology↗

bioRxiv2022

Exit from lag phase in populations of Pseudomonas fluorescens is determined by strong interactions among cells

The relationship between the number of cells colonizing a new environment and time for resumption of growth is a subject of long-standing interest. In microbiology this is known as the "inoculum effect". Its mechanistic basis is unclear with possible explanations ranging from the independent actions of individual cells, to collective actions of populations of cells. Progress requires precise measurement of lag time distributions while at the same time, experimentally controlling inoculum size. Here we use a millifluidic droplet device in which the growth dynamics of hundreds of populations founded by different numbers of Pseudomonas fluorescens cells, ranging from a single cell, to one thousand cells, were followed in real time. Our data show that lag phase decreases with inoculum size. The average decrease, variance across droplets, and distribution shapes, follow predictions of extreme value theory, where the inoculum lag time is determined by the minimum value sampled from the single-cell distribution. Our experimental results show that exit from lag phase depends on strong interactions among cells, consistent with a "leader-cell" triggering end of lag phase for the entire population.

microbiology↗

bioRxiv2021

Beyond Fitness Decoupling: Tradeoff-breaking during Evolutionary Transitions in Individuality

Evolutionary transitions in individuality (ETIs) involve the formation of Darwinian collectives from Darwinian particles. The transition from cells to multicellular life is a prime example. During an ETI, collectives become units of selection in their own right. However, the underlying processes are poorly understood. One observation used to identify the completion of an ETI is an increase in collective-level performance accompanied by a decrease in particle-level performance, for example measured by growth rate. This seemingly counterintuitive dynamic has been referred to as "fitness decoupling" and has been used to interpret both models and experimental data. Using a mathematical approach, we show this concept to be problematic in that the fitness of particles and collectives can never decouple--calculations of particle and collective fitness performed over appropriate and equivalent time intervals are necessarily the same. By way of solution, we draw attention to the value of mechanistic approaches that emphasise traits, and tradeoffs among traits, as opposed to fitness. This trait-based approach is sufficient to capture dynamics that underpin evolutionary transitions. In addition, drawing upon both experimental and theoretical studies, we show that while early stages of transitions might often involve tradeoffs among particle traits, later--and critical--stages are likely to involve the rupture of such tradeoffs. Thus, tradeoff-breaking stands as a useful marker for ETIs.

evolutionary biology↗

bioRxiv2021

Single-cell imaging of Pseudomonas reveals dynamic polar accumulation of the extracellular iron-scavenger pyoverdin

Pyoverdin is a water-soluble metal-chelator synthesized by members of the genus Pseudomonas and used for the acquisition of insoluble ferric iron. Although freely diffusible in aqueous environments, preferential dissemination of pyoverdin among adjacent cells, fine-tuning of intracellular siderophore concentrations, and fitness advantages to pyoverdin-producing versus nonproducing cells, indicate control of location and release. Here, using time-lapse fluorescence microscopy to track single cells in growing microcolonies of Pseudomonas fluorescens SBW25, we show accumulation of pyoverdin at cell poles. Accumulation occurs on cessation of cell growth, is achieved by cross-feeding in pyoverdin-nonproducing mutants and is reversible. Moreover, accumulation coincides with localization of a fluorescent periplasmic reporter, suggesting that pyoverdin accumulation at cell poles is part of the general cellular response to starvation. Compatible with this conclusion is absence of non-accumulating phenotypes in a range of pyoverdin mutants. Analysis of the performance of pyoverdin-producing and nonproducing cells under conditions promoting polar accumulation shows an advantage to accumulation on resumption of growth after stress. Examination of pyoverdin polar accumulation in a multispecies community and in a range of laboratory and natural species of Pseudomonas, including P. aeruginosa PAO1 and P. putida KT2440, confirms that the phenotype is characteristic of Pseudomonas.

microbiology↗

bioRxiv2021

REPINs are facultative genomic symbionts of bacterial genomes

Integrative mobile genetic elements (MGEs), such as transposons and insertion sequences, propagate within bacterial genomes, but persistence times in individual lineages are short. For long-term survival, MGEs must continuously invade new hosts by horizontal transfer. Theoretically, MGEs that persist for millions of years in single lineages, and are thus subject to vertical inheritance, should not exist. Here we draw attention to an exception -- a class of MGE termed REPIN. REPINs are non-autonomous MGEs whose duplication depends on non-jumping RAYT transposases. Comparisons of REPINs and typical MGEs show that replication rates of REPINs are orders of magnitude lower, REPIN population size fluctuations correlate with changes in available genome space, REPIN conservation depends on RAYT function, and REPIN diversity accumulates within host lineages. These data lead to the hypothesis that REPINs form enduring, beneficial associations with eubacterial chromosomes. Given replicative nesting, our hypothesis predicts conflicts arising from the diverging effects of selection acting simultaneously on REPINs and host genomes. Evidence in support comes from patterns of REPIN abundance and diversity in two distantly related bacterial species. Together this bolsters the conclusion that REPINs are the genetic counterpart of mutualistic endosymbiotic bacteria.

evolutionary biology↗

bioRxiv2021

Transposable elements drive the evolution of genome streamlining

Eukaryotes and prokaryotes have distinct genome architectures, with marked differences in genome size, the ratio of coding/non-coding DNA, and the abundance of transposable elements (TEs). As TEs replicate independently of their hosts, the proliferation of TEs is thought to have driven genome expansion in eukaryotes. However, prokaryotes also have TEs in intergenic spaces, so why do prokaryotes have small, streamlined genomes? Using an in silico model describing the genomes of single-celled asexual organisms that co-evolve with TEs, we show that TEs acquired from the environment by horizontal gene transfer can drive the evolution of genome streamlining. The process depends on local interactions and is underpinned by rock-paper-scissor dynamics in which populations of cells with streamlined genomes beat TEs, which beat non-streamlined genomes, in continuous and repeating cycles. Streamlining is maladaptive to individual cells, but delivers lineage-level benefits. Streamlining does not evolve in sexually reproducing populations because recombination partially frees TEs from the deleterious effects they cause.

evolutionary biology↗