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The evolution of complex life forms, exemplified by multicellular organisms, can be traced through a series of evolutionary transitions in individuality, beginning with the origin of life, followed by the emergence of the eukaryotic cell, and, among other transitions, culminating in the shift from unicellularity to multicellularity. Several attempts have been made to explain the origins of such transitions, many of which have been internalist (i.e., based largely on internal properties of ancestral entities). Here, we show how externalist perspectives can shed new light on questions pertaining to evolutionary transitions in individuality. We do this by presenting the ecological scaffolding framework in which properties of complex life forms arise from an external scaffold. Ultimately, we anticipate that progress will come from recognition of the importance of both the internalist and externalist modes of explanation. We illustrate this by considering an extension of the ecological scaffolding model in which cells modify the environment that later becomes the scaffold giving rise to multicellular individuality., Competing Interests: The authors declare no conflicts of interest., (© 2024 The Author(s). Ecology and Evolution published by John Wiley & Sons Ltd.)

Competing Interests: The authors declare no conflicts of interest.

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. Most ICEs characterized to date encode readily observable phenotypes contributing to symbiosis, pathogenicity, and antimicrobial resistance, yet the majority of ICEs carry genes of unknown function. Recent observations of 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. Fifty-three unique ICE types were identified across the P. syringae species complex. Together they form a distinct family of ICEs (PsICEs) that share a distant relationship to ICEs found in Pseudomonas aeruginosa . PsICEs are defined by conserved backbone genes punctuated by an array of accessory cargo genes, are highly recombinogenic, and display distinct evolutionary histories compared to their bacterial hosts. The most common cargo is a recently disseminated 16-kb mobile genetic element designated Tn 6212 . Deletion of Tn 6212 did not alter pathogen growth in planta, but mutants displayed fitness defects when grown on tricarboxylic acid (TCA) cycle intermediates. RNA-seq analysis of a set of nested deletion mutants showed that a Tn 6212 -encoded LysR regulator has global effects on chromosomal gene expression. We show that Tn 6212 responds to preferred carbon sources and manipulates bacterial metabolism to maximize growth., Competing Interests: Competing interests statement:The authors declare no competing interest.

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 ., Competing Interests: The authors declare no competing interests., (© The Author(s) 2024. Published by Oxford University Press on behalf of FEMS.)

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 approaches are necessary to increase our understanding beyond MGEs shaping individual populations, towards their impacts on complex microbial communities. A bioinformatic pipeline (xenoseq) was developed to cross-compare metagenomic samples from microbial consortia evolving in parallel, aimed at identifying MGE dissemination, which was applied to compost communities which underwent periodic mixing of MGEs. We show that xenoseq can distinguish movement of MGEs from demographic changes in community composition that otherwise confounds identification, and furthermore demonstrate the discovery of various unexpected entities. Of particular interest was a nanobacterium of the candidate phylum radiation (CPR) which is closely related to a species identified in groundwater ecosystems (Candidatus Saccharibacterium), and appears to have a parasitic lifestyle. We also highlight another prolific mobile element, a 313 kb plasmid hosted by a Cellvibrio lineage. The host was predicted to be capable of nitrogen fixation, and acquisition of the plasmid coincides with increased ammonia production. Taken together, our data show that new experimental strategies combined with bioinformatic analyses of metagenomic data stand to provide insight into the nanobiome as a driver of microbial community evolution., (© 2023. ISME Publications B.V.)

In recent years, evolutionary biologists have developed an 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., (© 2023. The Author(s).)

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, patch fitness increases but comes at a cost to cell growth rates. This creates conditions that initiate evolutionary transitions in individuality. A key feature of the scaffold is a bottleneck created during dispersal, causing patches to be 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 to investigate the effect of bottleneck size on the evolutionary dynamics of both cells and collectives. We show that larger bottlenecks simply slow the dynamics, but, at some point, which depends on the parameters of the within-patch model, the direction of evolution towards the equilibrium reverses. Introduction of random fluctuations in bottleneck sizes with some positive probability of smaller sizes counteracts this, even when the probability of smaller bottlenecks is minimal., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

That humans might undergo future evolutionary transitions in individuality (ETIs) seems fanciful. However, drawing upon recent thinking concerning the origins of properties that underpin ETIs, I argue that certain ETIs are imminently realizable. Central to my argument is recognition that heritable variance in fitness at higher levels of organization can be externally imposed (scaffolded) by specific ecological structures and cultural practices. While ETIs to eusociality seem highly improbable, ETIs involving symbioses between humans and artificial intelligence (AI) can be readily envisaged. A necessary requirement is that fitness-affecting interactions between humans and AI devices are inherited by offspring. The Mendelian nature of human reproduction ensures that offspring resemble parents. Reproduction of AI devices requires nothing more than transference of algorithms from parental AI devices to devices that are assigned to offspring. This simple copying, combined with societal structures that require humans to carry AI devices, ensures heritable variance in fitness at the level of both interacting partners. Selection at the collective level will drive alignment of replicative fates and increase co-dependency, thus alleviating need for continual imposition of externally imposed scaffolds. I conclude by drawing attention to the immediacy of such transitions and express concern over possibilities for malevolent manipulation. This article is part of the theme issue 'Human socio-cultural evolution in light of evolutionary transitions'.

We report a genome update for Pseudomonas fluorescens isolate SBW25. The updated genome assembly, which was derived from the original isolate, is based on PacBio long-read sequence data. It shows three minor differences, compared with the previously published genome sequence. Original annotations were merged with recent automated annotations to preserve information.

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., (© 2022 The Authors. BioEssays published by Wiley Periodicals LLC.)

Evolution has traditionally been a historical and descriptive science, and predicting future evolutionary processes has long been considered impossible. However, evolutionary predictions are increasingly being developed and used in medicine, agriculture, biotechnology and conservation biology. Evolutionary predictions may be used for different purposes, such as to prepare for the future, to try and change the course of evolution or to determine how well we understand evolutionary processes. Similarly, the exact aspect of the evolved population that we want to predict may also differ. For example, we could try to predict which genotype will dominate, the fitness of the population or the extinction probability of a population. In addition, there are many uses of evolutionary predictions that may not always be recognized as such. The main goal of this review is to increase awareness of methods and data in different research fields by showing the breadth of situations in which evolutionary predictions are made. We describe how diverse evolutionary predictions share a common structure described by the predictive scope, time scale and precision. Then, by using examples ranging from SARS-CoV2 and influenza to CRISPR-based gene drives and sustainable product formation in biotechnology, we discuss the methods for predicting evolution, the factors that affect predictability and how predictions can be used to prevent evolution in undesirable directions or to promote beneficial evolution (i.e. evolutionary control). We hope that this review will stimulate collaboration between fields by establishing a common language for evolutionary predictions., Competing Interests: The authors declare that there is no conflict of interest., (© 2022 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd.)

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. Here, we use a millifluidic droplet device in which the growth dynamics of hundreds of populations founded by controlled 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 decrease of average lag time and its variance across droplets, as well as lag time 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., Competing Interests: No competing interests are declared., (© The Author(s) 2022. Published by Oxford University Press on behalf of FEMS.)

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. Extending and unifying results from the literature, we show that fitness of particles and collectives can never decouple because calculations of fitness performed over appropriate and equivalent time intervals are necessarily the same provided the population reaches a stable collective size distribution. 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, when observed in the context of ETIs, tradeoff-breaking events stand as a useful marker of these transitions., Competing Interests: PB, GD, CR, KH No competing interests declared, PR Reviewing editor, eLife, (© 2022, Bourrat, Doulcier et al.)

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 coevolve with TEs, we show that TEs acquired from the environment by horizontal gene transfer can promote the evolution of genome streamlining. The process depends on local interactions and is underpinned by rock-paper-scissors dynamics in which populations of cells with streamlined genomes beat TEs, which beat non-streamlined genomes, which beat streamlined genomes, in continuous and repeating cycles. Streamlining is maladaptive to individual cells, but improves lineage viability by hindering the proliferation of TEs. Streamlining does not evolve in sexually reproducing populations because recombination partially frees TEs from the deleterious effects they cause. This article is part of the theme issue 'The secret lives of microbial mobile genetic elements'.

The function of microbes can be inferred from knowledge of genes specifically expressed in natural environments. Here, we report the in vivo transcriptome of the entomopathogenic bacterium Yersinia entomophaga MH96, captured during initial, septicemic, and pre-cadaveric stages of intrahemocoelic infection in Galleria mellonella. A total of 1285 genes were significantly upregulated by MH96 during infection; 829 genes responded to in vivo conditions during at least one stage of infection, 289 responded during two stages of infection, and 167 transcripts responded throughout all three stages of infection compared to in vitro conditions at equivalent cell densities. Genes upregulated during the earliest infection stage included components of the insecticidal toxin complex Yen-TC (chi1, chi2, and yenC1), genes for rearrangement hotspot element containing protein yenC3, cytolethal distending toxin cdtAB, and vegetative insecticidal toxin vip2. Genes more highly expressed throughout the infection cycle included the putative heat-stable enterotoxin yenT and three adhesins (usher-chaperone fimbria, filamentous hemagglutinin, and an AidA-like secreted adhesin). Clustering and functional enrichment of gene expression data also revealed expression of genes encoding type III and VI secretion system-associated effectors. Together these data provide insight into the pathobiology of MH96 and serve as an important resource supporting efforts to identify novel insecticidal agents., (© The Author(s) 2020. Published by Oxford University Press on behalf of Genetics Society of America.)

Interactions among microbial cells can generate new chemistries and functions, but exploitation requires establishment of communities that reliably recapitulate community-level phenotypes. Using mechanistic mathematical models, we show how simple manipulations to population structure can exogenously impose Darwinian-like properties on communities. Such scaffolding causes communities to participate directly in the process of evolution by natural selection and drives the evolution of cell-level interactions to the point where, despite underlying stochasticity, derived communities give rise to offspring communities that faithfully re-establish parental phenotype. The mechanism is akin to a developmental process ( developmental correction ) that arises from density-dependent interactions among cells. Knowledge of ecological factors affecting evolution of developmental correction has implications for understanding the evolutionary origin of major egalitarian transitions, symbioses, and for top-down engineering of microbial communities., Competing Interests: GD, AL, SD No competing interests declared, PR Reviewing editor, eLife and founding member of MilliDrop, (© 2020, Doulcier et al.)

The challenge of moving beyond descriptions of microbial community composition to the point where understanding underlying eco-evolutionary dynamics emerges is daunting. While it is tempting to simplify through use of model communities composed of a small number of types, there is a risk that such strategies fail to capture processes that might be specific and intrinsic to complexity of the community itself. Here, we describe approaches that embrace this complexity and show that, in combination with metagenomic strategies, dynamical insight is increasingly possible. Arising from these studies is mounting evidence of rapid eco-evolutionary change among lineages and a sense that processes, particularly those mediated by horizontal gene transfer, not only are integral to system function, but are central to long-term persistence. That such dynamic, systems-level insight is now possible, means that the study and manipulation of microbial communities can move to new levels of inquiry. This article is part of the theme issue 'Conceptual challenges in microbial community ecology'.

Microbial communities underpin the Earth's biological and geochemical processes, but their complexity hampers understanding. Motivated by the challenge of diversity and the need to forge ways of capturing dynamical behaviour connecting genes to function, biologically independent experimental communities comprising hundreds of microbial genera were established from garden compost and propagated on nitrogen-limited minimal medium with cellulose (paper) as sole carbon source. After 1 year of bi-weekly transfer, communities retained hundreds of genera. To connect genes to function, we used a simple experimental manipulation that involved the periodic collection of selfish genetic elements (SGEs) from separate communities, followed by pooling and redistribution across communities. The treatment was predicted to promote amplification and dissemination of SGEs and thus horizontal gene transfer. Confirmation came from comparative metagenomics, which showed the substantive movement of ecologically significant genes whose dynamic across space and time could be followed. Enrichment of genes implicated in nitrogen metabolism, and particularly ammonification, prompted biochemical assays that revealed a measurable impact on community function. Our simple experimental strategy offers a conceptually new approach for unravelling dynamical processes affecting microbial community function. This article is part of the theme issue 'Conceptual challenges in microbial community ecology'.

Fluorescent pseudomonads represent one of the largest groups of bacteria inhabiting the surfaces of plants, but their genetic composition in planta is poorly understood. Here, we examined the population structure and diversity of fluorescent pseudomonads isolated from sugar beet grown at two geographic locations (Oxford, United Kingdom and Auckland, New Zealand). To seek evidence for niche adaptation, bacteria were sampled from three types of leaves (immature, mature, and senescent) and then characterized using a combination of genotypic and phenotypic analysis. We first performed multilocus sequence analysis (MLSA) of three housekeeping genes ( gapA , gltA , and acnB ) in a total of 152 isolates (96 from Oxford, 56 from Auckland). The concatenated sequences were grouped into 81 sequence types and 22 distinct operational taxonomic units (OTUs). Significant levels of recombination were detected, particularly for the Oxford isolates (rate of recombination to mutation (r/m) = 5.23 for the whole population). Subsequent ancestral analysis performed in STRUCTURE found evidence of six ancestral populations, and their distributions significantly differed between Oxford and Auckland. Next, their ability to grow on 95 carbon sources was assessed using the Biolog™ GN2 microtiter plates. A distance matrix was generated from the raw growth data ( A 660 ) and subjected to multidimensional scaling (MDS) analysis. There was a significant correlation between substrate utilization profiles and MLSA genotypes. Both phenotypic and genotypic analyses indicated presence of a geographic structure for strains from Oxford and Auckland. Significant differences were also detected for MLSA genotypes between strains isolated from immature versus mature/senescent leaves. The fluorescent pseudomonads thus showed an ecotypic population structure, suggestive of adaptation to both geographic conditions and local plant niches., Competing Interests: The authors declare no conflict of interest., (© 2020 The Authors. Ecology and Evolution published by John Wiley & Sons Ltd.)

Evolutionary transitions in individuality are central to the emergence of biological complexity. Recent experiments provide glimpses of processes underpinning the transition from single cells to multicellular life and draw attention to the critical role of ecology. Here, we emphasize this ecological dimension and argue that its current absence from theoretical frameworks hampers development of general explanatory solutions. Using mechanistic mathematical models, we show how a minimal ecological structure comprising patchily distributed resources and between-patch dispersal can scaffold Darwinian-like properties on collectives of cells. This scaffolding causes cells to participate directly in the process of evolution by natural selection as if they were members of multicellular collectives, with collectives participating in a death-birth process arising from the interplay between the timing of dispersal events and the rate of resource use by cells. When this timescale is sufficiently long and new collectives are founded by single cells, collectives experience conditions that favour evolution of a reproductive division of labour. Together our simple model makes explicit key events in the major evolutionary transition to multicellularity. It also makes predictions concerning the life history of certain pathogens and serves as an ecological recipe for experimental realization of evolutionary transitions.

Cellulose-overproducing wrinkly spreader mutants of Pseudomonas fluorescens SBW25 have been the focus of much investigation, but conditions promoting the production of cellulose in ancestral strain SBW25 and its effects and consequences have escaped in-depth investigation through lack of an in vitro phenotype. Here, using a custom-built device, we reveal that in static broth microcosms, ancestral SBW25 encounters environmental signals at the air-liquid interface that activate, via three diguanylate cyclase-encoding pathways (Wsp, Aws, and Mws), production of cellulose. Secretion of the polymer at the meniscus leads to modification of the environment and growth of numerous microcolonies that extend from the surface. Accumulation of cellulose and associated microbial growth leads to Rayleigh-Taylor instability resulting in bioconvection and rapid transport of water-soluble products over tens of millimeters. Drawing upon data, we built a mathematical model that recapitulates experimental results and captures the interactions between biological, chemical and physical processes. IMPORTANCE This work reveals a hitherto unrecognized behavior that manifests at the air-liquid interface that depends on production of cellulose and hints at undiscovered dimensions to bacterial life at surfaces. Additionally, the study links activation of known diguanylate cyclase-encoding pathways to cellulose expression and to signals encountered at the meniscus. Further significance stems from recognition of the consequences of fluid instabilities arising from surface production of cellulose for transport of water-soluble products over large distances., (Copyright © 2019 Ardré et al.)

Observations of bacteria at the single-cell level have revealed many instances of phenotypic heterogeneity within otherwise clonal populations, but the selective causes, molecular bases, and broader ecological relevance remain poorly understood. In an earlier experiment in which the bacterium Pseudomonas fluorescens SBW25 was propagated under a selective regime that mimicked the host immune response, a genotype evolved that stochastically switched between capsulation states. The genetic cause was a mutation in carB that decreased the pyrimidine pool (and growth rate), lowering the activation threshold of a preexisting but hitherto unrecognized phenotypic switch. Genetic components surrounding bifurcation of UTP flux toward DNA/RNA or UDP-glucose (a precursor of colanic acid forming the capsules) were implicated as key components. Extending these molecular analyses-and based on a combination of genetics, transcriptomics, biochemistry, and mathematical modeling-we show that pyrimidine limitation triggers an increase in ribosome biosynthesis and that switching is caused by competition between ribosomes and CsrA/RsmA proteins for the mRNA transcript of a positively autoregulated activator of colanic acid biosynthesis. We additionally show that in the ancestral bacterium the switch is part of a program that determines stochastic entry into a semiquiescent capsulated state, ensures that such cells are provisioned with excess ribosomes, and enables provisioned cells to exit rapidly from stationary phase under permissive conditions., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Repeated evolution of functionally similar phenotypes is observed throughout the tree of life. The extent to which the underlying genetics are conserved remains an area of considerable interest. Previously, we reported the evolution of colony switching in two independent lineages of Pseudomonas fluorescens SBW25. The phenotypic and genotypic bases of colony switching in the first lineage (Line 1) have been described elsewhere. Here, we deconstruct the evolution of colony switching in the second lineage (Line 6). We show that, as for Line 1, Line 6 colony switching results from an increase in the expression of a colanic acid-like polymer (CAP). At the genetic level, nine mutations occur in Line 6. Only one of these-a nonsynonymous point mutation in the housekeeping sigma factor rpoD-is required for colony switching. In contrast, the genetic basis of colony switching in Line 1 is a mutation in the metabolic gene carB. A molecular model has recently been proposed whereby the carB mutation increases capsulation by redressing the intracellular balance of positive (ribosomes) and negative (RsmAE/CsrA) regulators of a positive feedback loop in capsule expression. We show that Line 6 colony switching is consistent with this model; the rpoD mutation generates an increase in ribosomal gene expression, and ultimately an increase in CAP expression., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Predicting evolutionary change poses numerous challenges. Here we take advantage of the model bacterium Pseudomonas fluorescens in which the genotype-to-phenotype map determining evolution of the adaptive 'wrinkly spreader' (WS) type is known. We present mathematical descriptions of three necessary regulatory pathways and use these to predict both the rate at which each mutational route is used and the expected mutational targets. To test predictions, mutation rates and targets were determined for each pathway. Unanticipated mutational hotspots caused experimental observations to depart from predictions but additional data led to refined models. A mismatch was observed between the spectra of WS-causing mutations obtained with and without selection due to low fitness of previously undetected WS-causing mutations. Our findings contribute toward the development of mechanistic models for forecasting evolution, highlight current limitations, and draw attention to challenges in predicting locus-specific mutational biases and fitness effects., Competing Interests: PL, EL, JH No competing interests declared, PR Reviewing editor, eLife, (© 2019, Lind et al.)

Pleurobranchaea maculata is a rarely studied species of the Heterobranchia found throughout the south and western Pacific-and recently recorded in Argentina-whose population genetic structure is unknown. Interest in the species was sparked in New Zealand following a series of dog deaths caused by ingestions of slugs containing high levels of the neurotoxin tetrodotoxin. Here we describe the genetic structure and demographic history of P. maculata populations from five principle locations in New Zealand based on extensive analyses of 12 microsatellite loci and the COI and CytB regions of mitochondrial DNA (mtDNA). Microsatellite data showed significant differentiation between northern and southern populations with population structure being associated with previously described regional variations in tetrodotoxin concentrations. However, mtDNA sequence data did not support such structure, revealing a star-shaped haplotype network with estimates of expansion time suggesting a population expansion in the Pleistocene era. Inclusion of publicly available mtDNA sequence sea slugs from Argentina did not alter the star-shaped network. We interpret our data as indicative of a single founding population that fragmented following geographical changes that brought about the present day north-south divide in New Zealand waters. Lack of evidence of cryptic species supports data indicating that differences in toxicity of individuals among regions are a consequence of differences in diet., Competing Interests: The authors have declared that no competing interests exist.

Interactions between commensal microbes and invading pathogens are understudied, despite their likely effects on pathogen population structure and infection processes. We describe the population structure and genetic diversity of a broad range of co-occurring Pseudomonas syringae isolated from infected and uninfected kiwifruit during an outbreak of bleeding canker disease caused by P. syringae pv. actinidiae (Psa) in New Zealand. Overall population structure was clonal and affected by ecological factors including infection status and cultivar. Most isolates are members of a new clade in phylogroup 3 (PG3a), also present on kiwifruit leaves in China and Japan. Stability of the polymorphism between pathogenic Psa and commensal P. syringae PG3a isolated from the same leaf was tested using reciprocal invasion from rare assays in vitro and in planta. P. syringae G33C (PG3a) inhibited Psa NZ54, while the presence of Psa NZ54 enhanced the growth of P. syringae G33C. This effect could not be attributed to virulence activity encoded by the Type 3 secretion system of Psa. Together our data contribute toward the development of an ecological perspective on the genetic structure of pathogen populations., (© 2018 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Over the last two decades interest in direct realisation of evolutionary process and the possibilities presented by real time evolution experiments with microbes have escalated. Long-term selection experiments with bacteria have made increasingly transparent the process of evolution by natural selection. In this short article we consider what next for the field and do so by highlighting two areas of interest: the genotype-to-phenotype map and the constraints it imposes on evolution, and studies on major evolutionary transitions and in particular the importance of selection working over more than one timescale. The latter we discuss in light of new technologies that allow imposition of Darwinian properties on populations and communities and how this allows exploration of new avenues of research. We conclude by commenting on microbial communities and the operation of evolutionary processes that are likely intrinsic-and specific-to communities., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Reproduction is a defining feature of living systems. To reproduce, aggregates of biological units (e.g., multicellular organisms or colonial bacteria) must fragment into smaller parts. Fragmentation modes in nature range from binary fission in bacteria to collective-level fragmentation and the production of unicellular propagules in multicellular organisms. Despite this apparent ubiquity, the adaptive significance of fragmentation modes has received little attention. Here, we develop a model in which groups arise from the division of single cells that do not separate but stay together until the moment of group fragmentation. We allow for all possible fragmentation patterns and calculate the population growth rate of each associated life cycle. Fragmentation modes that maximise growth rate comprise a restrictive set of patterns that include production of unicellular propagules and division into two similar size groups. Life cycles marked by single-cell bottlenecks maximise population growth rate under a wide range of conditions. This surprising result offers a new evolutionary explanation for the widespread occurrence of this mode of reproduction. All in all, our model provides a framework for exploring the adaptive significance of fragmentation modes and their associated life cycles.

Knowledge of adaptive processes encompasses understanding the emergence of new genes. Computational analyses of genomes suggest that new genes can arise by domain swapping; however, empirical evidence has been lacking. Here we describe a set of nine independent deletion mutations that arose during selection experiments with the bacterium Pseudomonas fluorescens in which the membrane-spanning domain of a fatty acid desaturase became translationally fused to a cytosolic di-guanylate cyclase, generating an adaptive 'wrinkly spreader' phenotype. Detailed genetic analysis of one gene fusion shows that the mutant phenotype is caused by relocalization of the di-guanylate cyclase domain to the cell membrane. The relative ease by which this new gene arose, along with its functional and regulatory effects, provides a glimpse of mutational events and their consequences that are likely to have a role in the evolution of new genes.

The two-component system CbrAB is the principal regulator for cellular metabolic balance in Pseudomonas fluorescens SBW25 and is necessary for growth on many substrates including xylose. To understand the regulatory linkage between CbrAB and genes for xylose utilization (xut), we performed transposon mutagenesis of ΔcbrB to select for Xut + suppressors. This led to identification of crc and hfq. Subsequent genetic and biochemical analysis showed that Crc and Hfq are key mediators of succinate-provoked carbon catabolite repression (CCR). Specifically, Crc/Hfq sequentially bind to mRNAs of both the transcriptional activator and structural genes involved in xylose catabolism. However, in the absence of succinate, repression is relieved through competitive binding by two ncRNAs, CrcY and CrcZ, whose expression is activated by CbrAB. These findings provoke a model for CCR in which it is assumed that crc and hfq are functionally complementary, whereas crcY and crcZ are genetically redundant. Inactivation of either crcY or crcZ produced no effects on bacterial fitness in laboratory media, however, results of mathematical modelling predict that the co-existence of crcY and crcZ requires separate functional identity. Finally, we provide empirical evidence that CCR is advantageous in nutrient-complex environments where preferred carbon sources are present at high concentrations but fluctuate in their availability., (© 2017 John Wiley & Sons Ltd.)

Selfish genetic elements, such as insertion sequences and transposons are found in most genomes. Transposons are usually identifiable by their high copy number within genomes. In contrast, REP-associated tyrosine transposases (RAYTs), a recently described class of bacterial transposase, are typically present at just one copy per genome. This suggests that RAYTs no longer copy themselves and thus they no longer function as a typical transposase. Motivated by this possibility we interrogated thousands of fully sequenced bacterial genomes in order to determine patterns of RAYT diversity, their distribution across chromosomes and accessory elements, and rate of duplication. RAYTs encompass exceptional diversity and are divisible into at least five distinct groups. They possess features more similar to housekeeping genes than insertion sequences, are predominantly vertically transmitted and have persisted through evolutionary time to the point where they are now found in 24% of all species for which at least one fully sequenced genome is available. Overall, the genomic distribution of RAYTs suggests that they have been coopted by host genomes to perform a function that benefits the host cell., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Cupins form one of the most functionally diverse superfamilies of proteins, with members performing a wide range of catalytic, non-catalytic, and regulatory functions. HutD is a predicted bicupin protein that is involved in histidine utilization (Hut) in Pseudomonas species. Previous genetic analyses have suggested that it limits the upper level of Hut pathway expression, but its mechanism of action is unknown. Here, we have determined the structure of PfluHutD at 1.74 Å resolution in several crystallization conditions, and identified N-formyl-l-glutamate (FG, a Hut pathway intermediate) as a potential ligand in vivo. Proteins 2017; 85:1580-1588. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

Recurring epidemics of kiwifruit (Actinidia spp.) bleeding canker disease are caused by Pseudomonas syringae pv. actinidiae (Psa). In order to strengthen understanding of population structure, phylogeography, and evolutionary dynamics, we isolated Pseudomonas from cultivated and wild kiwifruit across six provinces in China. Based on the analysis of 80 sequenced Psa genomes, we show that China is the origin of the pandemic lineage but that strain diversity in China is confined to just a single clade. In contrast, Korea and Japan harbor strains from multiple clades. Distinct independent transmission events marked introduction of the pandemic lineage into New Zealand, Chile, Europe, Korea, and Japan. Despite high similarity within the core genome and minimal impact of within-clade recombination, we observed extensive variation even within the single clade from which the global pandemic arose., (© The Author(s) 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Model microbial systems provide opportunity to understand the genetic bases of ecological traits, their evolution, regulation and fitness contributions. Experimental populations of Pseudomonas fluorescens rapidly diverge in spatially structured microcosms producing a range of surface-colonising forms. Despite divergent molecular routes, wrinkly spreader (WS) niche specialist types overproduce a cellulosic polymer allowing mat formation at the air-liquid interface and access to oxygen. Given the range of ways by which cells can form mats, such phenotypic parallelism is unexpected. We deleted the cellulose-encoding genes from the ancestral genotype and asked whether this mutant could converge on an alternate phenotypic solution. Two new traits were discovered. The first involved an exopolysaccharide encoded by pgaABCD that functions as cell-cell glue similar to cellulose. The second involved an activator of an amidase (nlpD) that when defective causes cell chaining. Both types form mats, but were less fit in competition with cellulose-based WS types. Surprisingly, diguanylate cyclases linked to cellulose overexpression underpinned evolution of poly-beta-1,6-N-acetyl-d-glucosamine (PGA)-based mats. This prompted genetic analyses of the relationships between the diguanylate cyclases WspR, AwsR and MwsR, and both cellulose and PGA. Our results suggest that c-di-GMP regulatory networks may have been shaped by evolution to accommodate loss and gain of exopolysaccharide modules facilitating adaptation to new environments.

Horizontal gene transfer can precipitate rapid evolutionary change. In 2010 the global pandemic of kiwifruit canker disease caused by Pseudomonas syringae pv. actinidiae (Psa) reached New Zealand. At the time of introduction, the single clone responsible for the outbreak was sensitive to copper, however, analysis of a sample of isolates taken in 2015 and 2016 showed that a quarter were copper resistant. Genome sequences of seven strains showed that copper resistance - comprising czc/cusABC and copABCD systems - along with resistance to arsenic and cadmium, was acquired via uptake of integrative conjugative elements (ICEs), but also plasmids. Comparative analysis showed ICEs to have a mosaic structure, with one being a tripartite arrangement of two different ICEs and a plasmid that were isolated in 1921 (USA), 1968 (NZ) and 1988 (Japan), from P. syringae pathogens of millet, wheat and kiwifruit respectively. Two of the Psa ICEs were nearly identical to two ICEs isolated from kiwifruit leaf colonists prior to the introduction of Psa into NZ. Additionally, we show ICE transfer in vitro and in planta, analyze fitness consequences of ICE carriage, capture the de novo formation of novel recombinant ICEs, and explore ICE host-range., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Oxidative stress is a major cause of mutation but little is known about how growth in the absence of oxygen impacts the rate and spectrum of mutations. We employed long-term mutation accumulation experiments to directly measure the rates and spectra of spontaneous mutation events in Escherichia coli populations propagated under aerobic and anaerobic conditions. To detect mutations, whole genome sequencing was coupled with methods of analysis sufficient to identify a broad range of mutational classes, including structural variants (SVs) generated by movement of repetitive elements. The anaerobically grown populations displayed a mutation rate nearly twice that of the aerobic populations, showed distinct asymmetric mutational strand biases, and greater insertion element activity. Consistent with mutation rate and spectra observations, genes for transposition and recombination repair associated with SVs were up-regulated during anaerobic growth. Together, these results define differences in mutational spectra affecting the evolution of facultative anaerobes., Competing Interests: The authors have declared that no competing interests exist.

Determining the phenotype and genotype of single cells is central to understand microbial evolution. DNA sequencing technologies allow the detection of mutants at high resolution, but similar approaches for phenotypic analyses are still lacking. We show that a drop-based millifluidic system enables the detection of heritable phenotypic changes in evolving bacterial populations. At time intervals, cells were sampled and individually compartmentalized in 100 nL drops. Growth through 15 generations was monitored using a fluorescent protein reporter. Amplification of heritable changes-via growth-over multiple generations yields phenotypically distinct clusters reflecting variation relevant for evolution. To demonstrate the utility of this approach, we follow the evolution of Escherichia coli populations during 30 days of starvation. Phenotypic diversity was observed to rapidly increase upon starvation with the emergence of heritable phenotypes. Mutations corresponding to each phenotypic class were identified by DNA sequencing. This scalable lineage-tracking technology opens the door to large-scale phenotyping methods with special utility for microbiology and microbial population biology.

Objectives: We aimed to examine the dynamics of Staphylococcus aureus nasal carriage in healthy adults., Method: Selected S. aureus strains isolated from weekly nasal swabs obtained from 122 healthy young adults over a 13 week period were spa typed., Results: The median duration of intermittent carriage was 4 weeks (IQR 2-6) and the median interval between episodes of carriage of different spa types was 3.5 weeks (IQR 2.25-4). 6/19 (32%) Persistent carriers were colonised with more than one spa type during the study, and in two persistent carriers a brief period of mixed colonisation with two spa types was observed. Even when the carriage strain changed, it was very rare for persistent carriers to have a period during which they were culture-negative (only 6/188 (3%) swabs submitted by persistent carriers failed to culture S. aureus)., Conclusions: Our results imply that at least every eight weeks a healthy young adult is exposed to S. aureus sufficient to cause a new episode of carriage among intermittent carriers. Persistent carriers are almost always colonised with S. aureus and over the course of a year there will be at least one replacement of the dominant strain., (Copyright © 2016 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)

The evolution of cooperation in group-structured populations has received much attention, but little is known about the effects of different modes of migration of individuals between groups. Here, we have incorporated four different modes of migration that differ in the degree of coordination among the individuals. For each mode of migration, we identify the set of multiplayer games in which the cooperative strategy has higher fixation probability than defection. The comparison shows that the set of games under which cooperation may evolve generally expands depending upon the degree of coordination among the migrating individuals. Weak altruism can evolve under all modes of individual migration, provided that the benefit to cost ratio is high enough. Strong altruism, however, evolves only if the mode of migration involves coordination of individual actions. Depending upon the migration frequency and degree of coordination among individuals, conditions that allow selection to work at the level of groups can be established., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Bacterial degradation of xylose is sequentially mediated by two enzymes - an isomerase (XutA) and a xylulokinase (XutB) - with xylulose as an intermediate. Pseudomonas fluorescens SBW25, though capable of growth on xylose as a sole carbon source, encodes only one degradative enzyme XutA at the xylose utilization (xut) locus. Here, using site-directed mutagenesis and transcriptional assays, we have identified two functional xylulokinase-encoding genes (xutB1 and xutB2) and further show that expression of xutB1 is specifically induced by xylose. Surprisingly, xylose-induced xutB1 expression is mediated by the mannitol-responsive regulator MtlR, using xylulose rather than xylose as the direct inducer. In contrast, expression of the xutA operon is regulated by XutR - a transcriptional activator of the AraC family - in a xylose-, xylulose- and ribose-dependent manner. Detailed genetic and biochemical analyses of XutR, including DNase I footprinting assays, suggest an unconventional model of XutR regulation that does not involve DNA-looping, a mechanism typically found for AraC-type regulators from enteric bacteria. XutR functions as a dimer and recognizes two inverted repeat sequences, but binding to one half site is weak thus requiring an inducer molecule such as xylose for activation., (© 2015 John Wiley & Sons Ltd.)

The International Pseudomonas aeruginosa Consortium is sequencing over 1000 genomes and building an analysis pipeline for the study of Pseudomonas genome evolution, antibiotic resistance and virulence genes. Metadata, including genomic and phenotypic data for each isolate of the collection, are available through the International Pseudomonas Consortium Database (http://ipcd.ibis.ulaval.ca/). Here, we present our strategy and the results that emerged from the analysis of the first 389 genomes. With as yet unmatched resolution, our results confirm that P. aeruginosa strains can be divided into three major groups that are further divided into subgroups, some not previously reported in the literature. We also provide the first snapshot of P. aeruginosa strain diversity with respect to antibiotic resistance. Our approach will allow us to draw potential links between environmental strains and those implicated in human and animal infections, understand how patients become infected and how the infection evolves over time as well as identify prognostic markers for better evidence-based decisions on patient care.

Unlabelled: CbrA is an atypical sensor kinase found in Pseudomonas. The autokinase domain is connected to a putative transporter of the sodium/solute symporter family (SSSF). CbrA functions together with its cognate response regulator, CbrB, and plays an important role in nutrient acquisition, including regulation of hut genes for the utilization of histidine and its derivative, urocanate. Here we report on the findings of a genetic and biochemical analysis of CbrA with a focus on the function of the putative transporter domain. The work was initiated with mutagenesis of histidine uptake-proficient strains to identify histidine-specific transport genes located outside the hut operon. Genes encoding transporters were not identified, but mutations were repeatedly found in cbrA. This, coupled with the findings of [(3)H]histidine transport assays and further mutagenesis, implicated CbrA in histidine uptake. In addition, mutations in different regions of the SSSF domain abolished signal transduction. Site-specific mutations were made at four conserved residues: W55 and G172 (SSSF domain), H766 (H box), and N876 (N box). The mutations W55G, G172H, and N876G compromised histidine transport but had minimal effects on signal transduction. The H766G mutation abolished both transport and signal transduction, but the capacity to transport histidine was restored upon complementation with a transport-defective allele of CbrA, most likely due to interdomain interactions. Our combined data implicate the SSSF domain of CbrA in histidine transport and suggest that transport is coupled to signal transduction., Importance: Nutrient acquisition in bacteria typically involves membrane-bound sensors that, via cognate response regulators, determine the activity of specific transporters. However, nutrient perception and uptake are often coupled processes. Thus, from a physiological perspective, it would make sense for systems that couple the process of signaling and transport within a single protein and where transport is itself the stimulus that precipitates signal transduction to have evolved. The CbrA regulator in Pseudomonas represents a unique type of sensor kinase whose autokinase domain is connected to a transporter domain. We present genetic and biochemical evidence that suggests that CbrA plays a dual role in histidine uptake and sensing and that transport is dependent on signal transduction., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Replicate populations of natural and experimental organisms often show evidence of parallel genetic evolution, but the causes are unclear. The wrinkly spreader morph of Pseudomonas fluorescens arises repeatedly during experimental evolution. The mutational causes reside exclusively within three pathways. By eliminating these, 13 new mutational pathways were discovered with the newly arising WS types having fitnesses similar to those arising from the commonly passaged routes. Our findings show that parallel genetic evolution is strongly biased by constraints and we reveal the genetic bases. From such knowledge, and in instances where new phenotypes arise via gene activation, we suggest a set of principles: evolution proceeds firstly via pathways subject to negative regulation, then via promoter mutations and gene fusions, and finally via activation by intragenic gain-of-function mutations. These principles inform evolutionary forecasting and have relevance to interpreting the diverse array of mutations associated with clinically identical instances of disease in humans.

Phenotype switching is commonly observed in nature. This prevalence has allowed the elucidation of a number of underlying molecular mechanisms. However, little is known about how phenotypic switches arise and function in their early evolutionary stages. The first opportunity to provide empirical insight was delivered by an experiment in which populations of the bacterium Pseudomonas fluorescens SBW25 evolved, de novo, the ability to switch between two colony phenotypes. Here we unravel the molecular mechanism behind colony switching, revealing how a single nucleotide change in a gene enmeshed in central metabolism (carB) generates such a striking phenotype. We show that colony switching is underpinned by ON/OFF expression of capsules consisting of a colanic acid-like polymer. We use molecular genetics, biochemical analyses, and experimental evolution to establish that capsule switching results from perturbation of the pyrimidine biosynthetic pathway. Of central importance is a bifurcation point at which uracil triphosphate is partitioned towards either nucleotide metabolism or polymer production. This bifurcation marks a cell-fate decision point whereby cells with relatively high pyrimidine levels favour nucleotide metabolism (capsule OFF), while cells with lower pyrimidine levels divert resources towards polymer biosynthesis (capsule ON). This decision point is present and functional in the wild-type strain. Finally, we present a simple mathematical model demonstrating that the molecular components of the decision point are capable of producing switching. Despite its simple mutational cause, the connection between genotype and phenotype is complex and multidimensional, offering a rare glimpse of how noise in regulatory networks can provide opportunity for evolution.