ECB_02826, ECB_02827, ECB_02828, yghD, yghE, ECB_02831, ECB_02832, ECB_02833 Ecb_02825

Keywords: ogrK, yegZ, ECB_01989, ECB_01990, ECB_01991, ECB_01992, ECB_01993

Large-scale DNA rearrangements, including inversions, amplifications, duplications, deletions, insertions, and transposition of mobile genetic elements, are major drivers of evolution and strongly impact on chromosome organization and expression, thereby altering organismal phenotypes. However, their long-term evolutionary dynamics and effects on organismal fitness are often unknown. We addressed these questions by using the longestrunning evolution experiment, during which twelve independent populations are propagated from a common E. coli ancestor in a glucose-limited environment for now over 60,000 generations (26 years). Most past studies have focused on point mutations and small InDels.

Using evolved clones sampled over time in all 12 populations, we characterized all largescale DNA rearrangements by using whole genome sequences and Whole Genome Mapping TM (i.e optical mapping). After 40,000 generations, we identified a total of 110 rearrangements including 82 deletions, 19 inversions and 9 duplications. Many chromosomal regions were repeatedly affected by similar rearrangements and, at least in one population, they occurred early in evolution when fitness increase was strong. Therefore, many rearrangements may be under positive selection. At the very least, these rearrangements strongly affected the structure of the chromosome during evolution.

At the molecular level, we showed that ~ 70% of all rearrangements occurred by recombination between Insertion Sequence (IS) elements, illustrating their importance in mediating genome plasticity. We therefore investigated the distribution and temporal dynamics of these small mobile genetic elements in all 12 populations. We showed that IS elements were strong contributors of the total mutations after 40,000 generations. In one population, they even represented about half of the total mutations and one IS type, IS150, revealed a strong 6-fold increase in copy number, accounting for the production of most of the rearrangements detected in this population. We showed that IS150 revealed a dynamic temporal behavior with a strong expansion followed by domestication by the host. By testing 2

Table of Figures

three evolutionary scenarios, we demonstrated that the IS150 expansion was related to a strong fitness increase conferred by the initial transposition events that occurred before 2000 generations. Later, between 20,000 and 40,000 generations, we measured a decreased transposition frequency, likely owing to a down regulation imposed by the host. Finally, and for the first time, we developed an evolution model of IS dynamics confirming that the IS expansion was related to a threshold number of initial IS beneficial insertions. All of our data showed that large-scale chromosomal rearrangements and IS elements have played an active role in the evolutionary outcomes after 40,000 generations of bacterial evolution.

Résumé

Les réarrangements d'ADN à grande échelle, tels que inversions, amplifications, duplications, délétions, insertions et transposition des éléments génétiques mobiles, sont des acteurs essentiels de l'évolution. Ils ont une forte incidence sur l'organisation et l'expression des chromosomes, ce qui affecte le phénotype des organismes. Toutefois, la dynamique de ces réarrangements au cours de l'évolution et leurs effets sur l'adaptation des organismes sont souvent inconnus. Nous avons abordé ces questions en utilisant la plus longue expérience d'évolution en cours. A partir d'un ancêtre commun d'Escherichia coli, douze populations indépendantes sont cultivées dans un milieu limité en glucose depuis plus de 60 000 générations, soit 26 ans. La plupart des études antérieures ont porté sur les mutations ponctuelles et les petites insertions et délétions (InDels). En utilisant des clones isolés au cours du temps dans ces 12 populations, nous avons caractérisé les réarrangements d'ADN à grande échelle à la fois par l'analyse des séquences de génomes et par cartographie optique.

A 40 000 générations, nous avons identifié 110 réarrangements parmi lesquelles 82 délétions, 19 inversions et 9 duplications. Plusieurs régions du chromosome ont été touchées à plusieurs reprises par le même type de réarrangements dans des populations indépendantes. Dans une des populations au moins, les réarrangements se sont produits au début de l'expérience d'évolution, au moment où l'augmentation de la valeur sélective est la plus élevée. Par conséquent, certains de ces réarrangements pourraient être bénéfiques dans ces conditions.

Même dans le cas contraire, nous avons montré que ces réarrangements affectaient fortement la structure du chromosome au cours de l'expérience d'évolution.

Au niveau moléculaire, nous avons montré que ~ 70% des réarrangements se produisent par recombinaison entre séquences d'insertion (IS), ce qui illustre l'importance de ces dernières dans la plasticité du génome. Nous avons donc caractérisé la distribution et la dynamique de ces petits éléments génétiques mobiles dans l'ensemble des 12 populations. Nous avons montré que les éléments IS ont fortement contribué à l'ensemble des mutations après 40 000 générations. Dans une population, les IS représentent même la moitié des mutations, et un des types d'IS, IS150, présente une forte prolifération avec 6 fois plus de copies à 40 000 générations, intervenant dans la plupart des réarrangements détectés dans cette population.

Nous avons montré une forte dynamique temporelle d'IS150, avec une forte expansion suivie d'une domestication par l'hôte. En testant trois scenarii évolutifs, nous avons démontré que l'expansion d'IS150 était liée à une forte augmentation de la valeur sélective conférée par les événements initiaux de transposition ayant eu lieu avant 2000 générations. Plus tard, entre 20 000 et 40 000 générations, nous avons mesuré une diminution de la fréquence de transposition, probablement en raison d'une régulation négative de la transposition imposée par l'hôte. Enfin, et pour la première fois, nous avons développé un modèle d'évolution de la dynamique des IS, qui confirme que leur expansion est liée à un nombre seuil d'insertions bénéfiques initiales. Ces résultats montrent que les réarrangements chromosomiques à grande échelle et les éléments IS ont joué un rôle actif dans la trajectoire évolutive au cours de 40 000 générations d'évolution bactérienne.

Objectives

As we strive to understand the world around us, the most fundamental question of how we came to be is often evoked. To elucidate the evolutionary processes that may have been involved during the geological ages, many experiments have been designed in laboratories with the aim of seeing evolution in action. Most of these experiments used organisms that reproduce quickly, are easily to manipulate and can be preserved as living fossils. One classical organism used for over 100 years in laboratories is Escherichia coli. We can manipulate it in many ways and a wealth of information is available about its biological properties. Therefore, it is not surprising that E. coli is used in many evolution experiments in which an ancestral strain of E. coli is propagated under defined conditions for various time lengths. Professor Richard Lenski initiated in 1988, 12 independent populations of E.coli, founded from a common ancestor, and propagated them since then by daily transfers in a specified environment (See Introduction: Fig 7). The evolution experiment now represents over 60,000 generations of evolution. Many studies have been performed with these populations to characterize the pace of evolution including both phenotypic and genomic changes. All the populations have evolved higher fitness, larger cell sizes and faster division rates. Most genetic studies have focused on specific mutations and on their fitness effect by moving them in ancestral or evolved genetic backgrounds. However, except for few cases, large-scale rearrangements, (deletions, duplications/amplifications, inversions, transposition of Insertion Sequence (IS) elements) have not been exhaustively investigated.

The objective of my Ph.D thesis was to investigate the evolutionary dynamics of chromosomal rearrangements during the long-term evolution experiment of Richard Lenski. This manuscript will be divided in three parts: first, an Introduction will give an overview of the current knowledge about DNA rearrangements and IS elements, and of the evolution experiment. The second part will give the main results of the thesis, and the third part will discuss them. The results of my work are included in two publications, one in revision in mBio and one that will be submitted in the next months. First, I focused on the extensive analysis of all the chromosomal rearrangements that occurred during 40,000 generations of the long-term evolution experiment. To reach this goal, we took advantage of the whole genome sequences and optical maps of evolved clones sampled at 40,000 generations from all 12 populations. The borders of some rearrangements were precisely checked with a PCR strategy. Many rearrangements have been identified, including their molecular mechanisms, and two lines of evidence (parallelism, occurrence early during evolution) suggested that some may be under positive selection.

The second part of the results will investigate the evolutionary dynamics of the main drivers of the identified chromosomal rearrangements: Insertion Sequence (IS) elements. These mobile genetic elements are involved in chromosomal reorganisation and we analysed their distribution and activity in all 12 populations. The IS dynamics is based on IS fingerprints, analysis of genome sequences and in vivo measurements of transposition and recombination frequencies. We also, and for the first time, developed an evolution model of IS dynamics.

Both the experiments and the model revealed that the dynamics of IS elements, including a strong expansion in one population, was driven by Darwinian selection whereupon the initial insertions of one type of IS elements in one population were strongly beneficial for the bacterial cells.

General Introduction

I. Preface

Life is incredibly diverse. There exists life in every environment that humans have looked at, from clams, mussels, and vestimentiferan worms thriving on chemosynthetic microbial living in the deepest oceans near hydrothermal vents [START_REF] Grassle | Hydrothermal vent animals: distribution and biology[END_REF] to over 17 different bacterial taxa found in the troposphere ( 8-15km) above the earth's surface [START_REF] Deleon-Rodriguez | Microbiome of the upper troposphere: species composition and prevalence, effects of tropical storms, and atmospheric implications[END_REF]. Not only have ecological niches been occupied but there is also a huge variety in the size and complexity of the forms organisms have incarnated. The smallest free living organism is the prokaryote, Mycoplasma genitalium, which has a genome of 580,070 base pairs and a total of 470 predicted coding regions [START_REF] Fraser | The minimal gene complement of Mycoplasma genitalium[END_REF]. On the other hand, the largest genome known belongs to the eukaryote, Loblolly Pine and spans 23.2 Gbp with a predicted 50,172 genes present [START_REF] Neale | Decoding the massive genome of loblolly pine using haploid DNA and novel assembly strategies[END_REF]. Not only does the size of genomes of organisms vary vastly but so does their biological complexity [START_REF] Adami | What is complexity?[END_REF]. The large variations give the ability for life to colonise every space shows the huge capability organisms have to adapt and to use a variety of available resources.

While we are more perceptible to large, easily observable organisms, it is in fact prokaryotes, comprising of the families Archea and Bacteria, which make up the majority of the individuals alive on the planet. A conservative estimate of prokaryote numbers puts them at 4-6 x 10 30 cells in total [START_REF] Whitman | Prokaryotes: the unseen majority[END_REF] but the actual count is likely to be much higher. The number of species this represents is almost impossible to say as the species boundaries are not clearly defined for prokaryotes nor do we have the capacity to identify all the species. But a rough estimate gives approximately 2 × 10 6 species present in the sea, while a ton of soil could contain up to 4 × 10 6 different taxa [START_REF] Curtis | Estimating prokaryotic diversity and its limits[END_REF]. This huge number of individuals represents a large pool from which there can be adaptation and colonisation of new niches.

Not only do prokaryotes account for the highest number of cells but they are also the most rapidly replicating organisms found on earth. The fastest replicating bacteria can replicate once every 10 minutes given the right conditions [START_REF] Elsgaarda | Hydrothermal vents in Lake Tanganyika harbor spore-forming thermophiles with extremely rapid growth[END_REF]. This is close to the fastest possible duplication within the realms of thermodynamics [START_REF] England | Statistical physics of self-replication[END_REF]. On the other hand, eukaryotic organisms have a much slower duplication time, as fast dividing human cell may divided in about 24 hours, this may be due to an increase in eukaryotic physical complexity. With the number of cells combined with the capacity for fast growth, prokaryotes have a large potential to create genetic diversity and therefore be capable of colonising new ecological niches.

Generation of Diversity

According to classical theories all life descends from a common ancestor. When we observe the world around us there is, however, a huge variety in the forms that have evolved.

Therefore, to understand how these organisms have appeared we must understand the creation of the diversity. The creation and the selection of difference within a population allow it to adapt to new environments [START_REF] Nevo | Evolution of genome-phenome diversity under environmental stress[END_REF][START_REF] Sahney | Links between global taxonomic diversity, ecological diversity and the expansion of vertebrates on land[END_REF], to overcome both external and internal stresses and to be more robust to changes. This process occurs due to the selection of individuals within the population that are more adapted to a specific environment, therefore more likely to produce offspring. The process is best described by Darwin "As many more individuals of each species are born than can possibly survive; and as, consequently, there is a frequently recurring struggle for existence, it follows that any being, if it vary however slightly in any manner profitable to itself, under the complex and sometimes varying conditions of life, will have a better chance of surviving, and thus be naturally selected. From the strong principle of inheritance, any selected variety will tend to propagate its new and modified form." [START_REF] Darwin | On the Origin of Species[END_REF]. The creation of diversity comes about by changes to the genetic makeup of individuals within a population so to truly understand the apparition of diversity we must understand the mechanisms by which they appear.

Single nucleotide polymorphisms

The most common way to create genetic diversity is through point mutations generated by imperfect DNA replication. This happens at a rate of between 10 -7 and 10 -8 mutations per base pair duplicated in Escherichia coli [START_REF] Schaaper | Base selection, proofreading, and mismatch repair during DNA replication in Escherichia coli[END_REF]. This means that on average every two division cycles of E. coli will produce one mutation. Estimates for mutation rates in humans give numbers in the same order of magnitude [START_REF] Crow | How much do we know about spontaneous human mutation rates?[END_REF], however due to larger genome (6.4 x10 9 ) this translates to 64 mutations per zygote. Given this high rate of occurrence and ease of identification a lot of attention has been focused on SNP production and effects. However, , we know SNPs are not the only way by which genetic diversity can occur and the advent of modern biological techniques allows to illustrate more in depth these variations. Other less studied mutations in bacteria include chromosome rearrangements and insertion sequence (IS) movements.

II. Rearrangements

Large-scale genomic rearrangements play a major role in the evolution of species, both eukaryote and prokaryote [START_REF] Putnam | The amphioxus genome and the evolution of the chordate karyotype[END_REF][START_REF] Toussaint | Prokaryote genome fluidity: toward a system approach of the mobilome[END_REF] as they can generate effects not producible by SNP including gene and regulatory sequence reassortment, elimination of DNA, creation of duplications and evolution of new genes (Moore 2012). Large-scale DNA rearrangements occur spontaneously at low frequencies. For instance, duplication events arise at frequencies comprised between 10 -2 and 10 -5 duplication per cell division depending on their chromosomal location (Anderson and Roth 1981). The relatively low frequency of chromosome rearrangements can be explained by several factors.

First, culturing of strains may induce the loss of unstable DNA rearrangements like duplications, resulting in underestimation of their frequency [START_REF] Maharjan | A case of adaptation through a mutation in a tandem duplication during experimental evolution in Escherichia coli[END_REF].

Second, these previous estimates have all been performed based on observable phenotypes that were produced after the rearrangements occurred (Zieg and Kushner 1977;[START_REF] Albertini | On the formation of spontaneous deletions: the importance of short sequence homologies in the generation of large deletions[END_REF][START_REF] Segall | Rearrangement of the bacterial chromosome: forbidden inversions[END_REF][START_REF] Bierne | uvrD mutations enhance tandem repeat deletion in the Escherichia coli chromosome via SOS induction of the RecF recombination pathway[END_REF]. Third duplications are inherently unstable and they are prone to reversion that limits the estimation of their rate (Anderson and Roth 1981).

Mechanisms for rearrangements.

Chromosomal rearrangements will occasionally occur during the DNA replication or repair, and will involve DNA recombination. There are several documented methods of genome recombination. The most frequent being homologous recombination between two DNA segments sharing homology. Even though bacteria do not have many homologous genes, recombination occurs between ribosomal operons (Anderson and Roth 1981) and mobile genetic elements like transposons [START_REF] Cui | Coordinated phenotype switching with large-scale chromosome flip-flop inversion observed in bacteria[END_REF], insertion sequence (IS) elements [START_REF] Daveran-Mingot | A natural large chromosomal inversion in Lactococcus lactis is mediated by homologous recombination between two insertion sequences[END_REF], and prophage sequences (Iguchi 2006). Moreover there are cases of atypical rearrangements which involve homologous sequences [START_REF] Roth | Relative rates of homologous and nonhomologous recombination in transfected DNA[END_REF][START_REF] Weller | Identification of a DNA nonhomologous end-joining complex in bacteria[END_REF]. In all cases, homologous recombination requires a double strand break in the DNA and reparation of this break in a new conformation. Two main pathways have been described for bacterial homologous recombination. In both case, the main actor is RecA, a 38kDa protein, ubiquitous in all class of life. RecA is a DNA dependent ATPase and will bind to single strand DNA as a nucleoprotein filament [START_REF] Patel | A new model for SOS-induced mutagenesis: how RecA protein activates DNA polymerase V[END_REF]. In E. coli, RecA is involved in 2 cellular functions; one is recombinational DNA repair with the binding of single strand DNA and the strand invasion of the homologous DNA molecule. RecA is vital for cells as not only does it contribute to homologous recombination but it plays a vital role in restarting collapsed DNA replication forks [START_REF] Cox | The importance of repairing stalled replication forks[END_REF]. The second main function is biding to and promoting the self-cleavage of LexA, UmuD and polV to promote the SOS response. [START_REF] Michel | Recombination proteins and rescue of arrested replication forks[END_REF].

Depending of the nature of the DNA degradation, a single strand break (ssDNA) or a double strand break (dsDNA) molecular complexes RecFOR with the ssDNA or RecBCD with dsDNA will bind to the DNA partially degrade a part of the damaged molecule, load the RecA protein on the resulting single strand DNA molecule. The RecA filament will invade the homologous dsDNA and the resulting Holliday junction will migrated under the catalysis of RuvAB and RecG [START_REF] Michel | Recombination proteins and rescue of arrested replication forks[END_REF] (Fig 1). Non homologous end joining also occurs in bacteria and is RecA independent. In some bacteria as Mycobacteria and Bacillus subtilis, the enzymes involved in the rudimental bacterial NHEJ machinery consists of two proteins, namely the multifunctional ATP-dependent ligase-D and Ku [START_REF] Della | Mycobacterial Ku and ligase proteins constitute a two-component NHEJ repair machine[END_REF]). However, in E coli, Ku-like and ligase-D like homologue have not been detected and an alternative End-Joining mechanism involving the multiprotein complex RecBCD had been described [START_REF] Chayot | An end-joining repair mechanism in Escherichia coli[END_REF]. There are other types of recombination that are mediated by other methods than the main RecA pathway. Transposition of specific sequences such as IS elements are a prime example and are discussed in length later on.

Types of rearrangements and their effects

Two classes of rearrangements are described I) Incorporation of foreign DNA acquired by either horizontal transfer or through DNA scavenging and II) rearrangements of existing genes.

I .

II .

III .

IV.

V.

Horizontal gene transfer (Incorporation of foreign DNA)

Incorporation of foreign DNA is a major pathway for new gene acquisition in bacteria. This is especially important in the dissemination of antibacterial resistance genes throughout an ecosystem (Skippington and Ragan 2011). There are three main ways new DNA can be incorporated into the cell [START_REF] Halary | Network analyses structure genetic diversity in independent genetic worlds[END_REF], the first and most common is through conjugative transfer where there is a physical contact between two cells and DNA is exchanged through the gap that is created through both cell membranes. The main type of genetic material to be exchanged is F-plasmids that contain all the genes necessary for its own replication and sometimes can also contain gene coding for antibacterial properties. The donor and recipients are generally closely related species or found within the same order, but it can also occur between widely different species, for example between bacteria and fungi [START_REF] Zhang | The mycosphere constitutes an arena for horizontal gene transfer with strong evolutionary implications for bacterial-fungal interactions[END_REF]). This has a large impact on the evolution of both organisms as it gives them access to genes not found within their own pangenome [START_REF] Zhang | The mycosphere constitutes an arena for horizontal gene transfer with strong evolutionary implications for bacterial-fungal interactions[END_REF]. This method of genome rearrangement is very well studied due to its prevalence in evolution and dissemination of antibacterial resistance genes that can be carried by pathogenic islands on the transferred genetic material (for review see [START_REF] Schmidt | Pathogenicity islands in bacterial pathogenesis[END_REF]. Examples of resistance acquisition due to conjugation is frequently described (for reviews see [START_REF] Alekshun | Molecular mechanisms of antibacterial multidrug resistance[END_REF][START_REF] Acar | Antimicrobial resistance: a complex issue[END_REF] and occur in almost any environment where there is selection for resistance including in many animals [START_REF] Ahmed | Antimicrobial resistance in equine faecal Escherichia coli isolates from North West England[END_REF][START_REF] Pan | A multidrug-resistance region containing blaCTX-M-65, fosA3 and rmtB on conjugative IncFII plasmids in Escherichia coli ST117 isolates from chicken[END_REF]. Samples taken throughout nature also reveal that resistance carrying plasmids are widely spread in most environments and therefore constitute a very easily accessible pool of genes (for review see [START_REF] Allen | Call of the wild: antibiotic resistance genes in natural environments[END_REF]. Conjugation is also involved in the formation of social structures in biofilms and spreading of co-operative traits [START_REF] Ghigo | Natural conjugative plasmids induce bacterial biofilm development[END_REF][START_REF] Nogueira | Horizontal gene transfer of the secretome drives the evolution of bacterial cooperation and virulence[END_REF].

A second type of horizontal transfer is transformation, where a recipient can take up free DNA from the environment. There is some debate as to how the trait evolved. There is a high relative fitness cost for competent bacteria, due to associated protein synthesis and DNA length augmentation, which might explain why not all bacteria are competent [START_REF] Peterson | Gene expression analysis of the Streptococcus pneumoniae competence regulons by use of DNA microarrays[END_REF]. Therefore, in naturally competent cells there is a strict and intricate regulation of the many genes required (over 40 in Bacillus subtilis) [START_REF] Hamoen | Controlling competence in Bacillus subtilis: shared use of regulators[END_REF]. In general, DNA uptake in competent bacteria requires the presence of type IV pili or pseudo pili [START_REF] Chen | A macromolecular complex formed by a pilin-like protein in competent Bacillus subtilis[END_REF]. The large amount of DNA found in the environment could confer a number of new functions to bacteria. These include the acquisition of genes for resistance, biofilm and other beneficial aspects. Competence is also a way for acquisition of undamaged nucleotides. This in some part mitigates the effect of DNA damaging antimicrobials, especially antimicrobials creating double strand breaks. S. pneumoniae transformation protects against the bactericidal effect of mitomycin C and streptomycin [START_REF] Engelmoer | Competence increases survival during stress in Streptococcus pneumoniae[END_REF]. It is shown that most antibacterial treatments that produce double strand breaks in bacteria can induce competence in bacteria that are lacking the SOS-RecA response mechanism; E.coli has this mechanism and is therefore not naturally competent [START_REF] Charpentier | Antibiotics and UV radiation induce competence for natural transformation in Legionella pneumophila[END_REF][START_REF] Charpentier | Induction of competence for genetic transformation by antibiotics: convergent evolution of stress responses in distant bacterial species lacking SOS?[END_REF]. Giving argument that competence is an adaptation to reduce stress on the genome.

A third type of rearrangement involving foreign DNA is transduction. This is the incorporation of a viral genome into the host's. The viral DNA might carry additional genes that confer an advantage to the host. This form of horizontal transfer has the advantage that DNA is protected from DNAses in the environment; however it has a big cost to the cell as it is infected by a phage which has more potential to damage the cell. The most common example that is routinely used in laboratory settings is the lambda phage of E.coli. While it is a lytic bacteriophage, lambda phage can undergo a lysogenic phase wherein the phage DNA is incorporated into the host genome and then virulence factors are down regulated, leading to the phage maintaining itself in the genome and are subsequently included in DNA replication.

The insertion is site specific and the location of the site suggests that there has been coevolution between lambda phage and E.coli to facilitate this interaction [START_REF] Tal | Location of the unique integration site on an Escherichia coli chromosome by bacteriophage lambda DNA in vivo[END_REF]. While most of the literature focuses on lytic phages there are a large number of examples of temperate phages (for review see Fortier and Sekulovic 2013) Lysogenic phages can bring in new genes to the genome including virulence factors and can convert a nonpathogenic bacteria to a pathogenic one [START_REF] Waldor | Lysogenic conversion by a filamentous phage encoding cholera toxin[END_REF]. In addition to the transfer of virulence genes, prophages allow bacteria to adapt to rapidly changing environments. For example, they are important for gene expression in the various stages in the complicated life cycle of Bacillus anthracis [START_REF] Schuch | The secret life of the anthrax agent Bacillus anthracis: bacteriophage-mediated ecological adaptations[END_REF], for the biofilm formation [START_REF] Stanley | Environmental signals and regulatory pathways that influence biofilm formation[END_REF] and can be responsible for spore formation in Bacillus subtilis [START_REF] Silver-Mysliwiec | Bacteriophage-enhanced sporulation: comparison of spore-converting bacteriophages PMB12 and SP10[END_REF]. Since prophages are principally involved in adaptation to new environments, one can theorise that in a stable conditions, there might be a loss or attenuation of the dissemination of prophages.

Even though horizontal transfer is an important means of DNA rearrangements, there are organisms that are not naturally competent and cannot incorporate foreign DNA into their genomes. Many do this to limit the uptake of pathogens and DNA is broken down within the cells. However, organisms are able to increase their genetic diversity by the modification of their existing chromosome, rearrangement of genetic material has been documented in organisms representing eukaryotic, bacterial and archean domains of life.

Rearrangement of existing genome

Rearrangements of the existing genome play a major role in evolution. This can be in the form of deletions, inversions, duplications and amplifications of parts of the genome (Fig 2 ).

Identification of rearrangements

Chromosomal rearrangements are usually investigated by using methods such as pulsed-field gel electrophoresis, microarray-based hybridization experiments, and whole genome sequencing. Unfortunately, typical whole genome sequencing technologies that rely on sequencing short DNA fragment libraries cannot detect all large-scale DNA rearrangements, in particular inversions and other rearrangements involving long sequence repeats. Optical mapping techniques have been developed that produce a high-resolution ordered restriction map of bacterial genomes that can complement sequencing data (Fig. 3) [START_REF] Schwartz | Ordered restriction maps of Saccharomyces cerevisiae chromosomes constructed by optical mapping[END_REF][START_REF] Kotewicz | Optical maps distinguish individual strains of Escherichia coli O157 : H7[END_REF][START_REF] Shukla | Optical mapping reveals a large genetic inversion between two methicillin-resistant Staphylococcus aureus strains[END_REF][START_REF] Turner | Optical mapping and sequencing of the Escherichia coli KO11 genome reveal extensive chromosomal rearrangements, and multiple tandem copies of the Zymomonas mobilis pdc and adhB genes[END_REF].

Deletions

Deletions are the most commonly observed rearrangements observed in evolving populations and perform a versatile role in the evolution and the adaptation of populations to their environment [START_REF] Mira | Deletional bias and the evolution of bacterial genomes[END_REF]. They may cause massive gene loss and are important for the evacuation of superfluous genes that are not expressed in certain conditions [START_REF] Andersson | Genome degradation is an ongoing process in Rickettsia[END_REF].

The extra genes are therefore a cost to the organism as it increases the time and energy required for duplication and there may be residual transcription of unneeded proteins.

Obligate endosymbiont are a particular example as they show that with a massive streamlining of the genome they evacuate genes. Up to now, the smallest known genome is attributed to Nasuia deltocephalinicola an endosymbionte from sap feeding insect, with a 112 kb genome carrying only 137 protein encoding genes [START_REF] Bennett | Small, smaller, smallest: the origins and evolution of ancient dual symbioses in a Phloem-feeding insect[END_REF]. This represents an extreme in genome reduction. Genome reduction is also observed in the adaptation to any stable environment. In cystic fibrosis patients, deletions play an important part in the long-term evolution of P. aeruginosa, with some isolates losing up to 8% of its genome by homologous and non-legitimate deletions [START_REF] Rau | Deletion and acquisition of genomic content during early stage adaptation of Pseudomonas aeruginosa to a human host environment[END_REF].

Salmonella enterica cultured in rich media over 1000 generations showed significant levels of selection driven gene loss. Rearrangements occurred between Tn10 elements interspersed over the genome with some regions being more targeted. This further suggests that deletions are a way of evacuating superfluous DNA [START_REF] Koskiniemi | Selection-driven gene loss in bacteria[END_REF]. Deletions of specific gene targets are also a common feature during evolution including the emergence of resistance to antimicrobial peptides in E.coli where a deletion of a nonessential gene renders the bacteria resistant to a multi target peptide [START_REF] Narayanan | Mechanism of Escherichia coli Resistance to Pyrrhocoricin[END_REF]. Deletions can sometimes even be development-regulators, by creating chimeric genes involved in sporulation [START_REF] Abe | Regulated DNA rearrangement during sporulation in Bacillus weihenstephanensis KBAB4[END_REF]. In this case, a 48 kb element of the genome is excised from Bacillus subtilis which leads to the formation of a functional regulator sigK [START_REF] Abe | Regulated DNA rearrangement during sporulation in Bacillus weihenstephanensis KBAB4[END_REF].

Inversions

Inversions occur when a region of the chromosome is cut out of the chromosome by double strand breaks and then reinserted with the opposite orientation. Comparisons of genomes from closely related bacterial species have detected a high level of DNA inversions among chromosomal rearrangements [START_REF] Eisen | Evidence for symmetric chromosomal inversions around the replication origin in bacteria[END_REF]Tillier and Collins 2000;[START_REF] Zivanovic | Pyrococcus genome comparison evidences chromosome shuffling-driven evolution[END_REF]. Inversions play several roles in the genome. First it might change gene expression by either breaking the genes at the end points of the inversion, or by changing the orientation of genes in relation to the replication fork. In effect, genes that are transcribed in greater quantities are in the same direction as the replication fork on the leading strand, as this minimises the incidences of collisions between the RNA and the DNA polymerases (Rocha and Danchin 2003). Second inversions change the genome conformation by displacing secondary structures and by displacing genome features involved with physical chromosome conformation such as the parS site [START_REF] Umbarger | The three-dimensional architecture of a bacterial genome and its alteration by genetic perturbation[END_REF]). However, some genome features are not largely impacted since a selection bias exists leading to the over representation of inversions around the replication origin in bacteria that maintains the symmetry of the genome [START_REF] Eisen | Evidence for symmetric chromosomal inversions around the replication origin in bacteria[END_REF][START_REF] Darling | Dynamics of genome rearrangement in bacterial populations[END_REF].

The actual benefit of inversions is hard to quantify but they are present in a large number of species and can happen very easily. After sub culturing E. coli O157:H7 for only 50 passages on new media, one third of the isolated clones were found to have novel inversions (Iguchi A 2006). The comparison of Vibrio cholerae, Streptococcus pneumoniae and Mycobacterium tuberculosis to those of closely related species -E. coli, Streptococcus pyogenes and Mycobacterium leprae reveals that inversions (up to 10% of the genome) happen on a large scale during evolution, especially around the origin and terminus of replication [START_REF] Eisen | Evidence for symmetric chromosomal inversions around the replication origin in bacteria[END_REF]. A similar pattern can be found in Pseudomonas stutzeri [START_REF] Ginard | Genome organization of Pseudomonas stutzeri and resulting taxonomic and evolutionary considerations[END_REF], Pseudomonas aeruginosa [START_REF] Kresse | Impact of large chromosomal inversions on the adaptation and evolution of Pseudomonas aeruginosa chronically colonizing cystic fibrosis lungs[END_REF], Francisella tularensis [START_REF] Rohmer | Comparison of Francisella tularensis genomes reveals evolutionary events associated with the emergence of human pathogenic strains[END_REF], Neisseria gonorrhoeae [START_REF] Spencer-Smith | Sequence features contributing to chromosomal rearrangements in Neisseria gonorrhoeae[END_REF], Lactococcus lactis [START_REF] Daveran-Mingot | A natural large chromosomal inversion in Lactococcus lactis is mediated by homologous recombination between two insertion sequences[END_REF], and Staphylococcus aureus [START_REF] Cui | Coordinated phenotype switching with large-scale chromosome flip-flop inversion observed in bacteria[END_REF] when they are all compared within their respective pangenomes. On average 20 % of the genome is inverted in these cases.

Duplications and Amplifications

Duplications and amplification events are among the most important rearrangements to occur in asexual bacteria. Duplications is defined by a copying of existing genome while an amplification can result in more than two copies of a stretch of genome being present. The advent of mass genome sequencing of bacterial genomes reveals that the majority of duplications are small scale duplications such as tandem and operon duplications [START_REF] Gevers | Gene duplication and biased functional retention of paralogs in bacterial genomes[END_REF] A variable gene dosage caused by duplications allows organisms in overcoming limitations linked to inefficient gene expression as it increases the gene product. For example, in helping an evolving population to overcome limited expressions of specific functions such as to increase lactose uptake in lactose limited environments in E.coli [START_REF] Novick | Hyper-production of beta-galactosidase by Escherichia coli bacteria[END_REF], escape from certain stringent conditions by amplification of specific genes followed by mutations in defective genes [START_REF] Pranting | Escape from growth restriction in small colony variants of Salmonella typhimurium by gene amplification and mutation[END_REF], modification of gene expression [START_REF] Kugelberg | Multiple pathways of selected gene amplification during adaptive mutation[END_REF] or even contributes to antibiotic or metal resistance (Sun, Berg et al. 2009, von Rozycki et al. 2009). The temporary nature of duplications allows organisms to revert to their original state once the selection pressure is released (for review see [START_REF] Kondrashov | Gene duplication as a mechanism of genomic adaptation to a changing environment[END_REF]. Duplications can also have negative effects on the organism due to superfluous gene expression. This is particularly the case in eukaryote cells and is linked to diseases such as defective eye formation in Drosophila, [START_REF] Bridges | The Bar "Gene" a Duplication[END_REF], causes unbalance in protein complexes that are toxic in Saccharomyces cerevisiae [START_REF] Papp | Dosage sensitivity and the evolution of gene families in yeast[END_REF] or even is linked to autism in humans [START_REF] Sebat | Strong association of de novo copy number mutations with autism[END_REF]. In bacterial genomes even mildly deleterious effects of gene duplication are rapidly purged from the population.

Not only do duplications have an immediate impact on cells through altered gene dosage but there are many long term effects associated. Duplicated genes can act as a redundancy mechanism whereby if one gene is affected by a deleterious mutation there is always another functional copy [START_REF] Clark | Invasion and maintenance of a gene duplication[END_REF]. The long term fate for the duplicates is not fixed and it largely depends on the fitness change brought by the duplication. The most common fate for duplicated genes is to accumulate a number of mutations that inactivates them and creates pseudo genes from the duplicates [START_REF] Lynch | The evolutionary fate and consequences of duplicate genes[END_REF]. This fate, while common in eukaryotes, is less common in bacteria due to the cost increase in duplicating non-coding DNA. A second fate for gene duplication is for the distribution of the gene function over several genes. Through observation of the genomes of eukaryote organism it is clear that this model explains the phenomena of complementary of gene functions [START_REF] Force | Preservation of duplicate genes by complementary, degenerative mutations[END_REF]. This method of gene dissemination is also present in most sequenced bacteria and is true for around 89% of all homologous gene families with the other 11% accounted for by horizontal gene transfer [START_REF] Pushker | Comparative genomics of gene-family size in closely related bacteria[END_REF]). Finally, one of the duplicates can be subject to a rare beneficial mutation that endows it with a novel gene function while the remaining copy retains its ancestral function [START_REF] Nasvall | Real-time evolution of new genes by innovation, amplification, and divergence[END_REF]. This method of de novo gene creation is especially relevant in asexual species where there is no exchange of DNA. As many genes have secondary gene functions, duplication of a gene can favour this secondary gene product in a specific environment and therefore the duplication is maintained due to the secondary function rather than the primary function. A mutation might occur in one of the gene copies that might amplify the effect of a secondary gene function or the loss of the primary gene function and through gradual genetic drift the copies might diverge to the point where both genes have different functions [START_REF] Lynch | The probability of duplicate gene preservation by subfunctionalization[END_REF][START_REF] Ward | Subfunctionalization: How often does it occur? How long does it take?[END_REF]Elliot 2012). Another, less appreciated function of duplications is gene shuffling, similar to shuffling produced by inversions, where regulatory networks of genes can be changed. This can be due to promoter capture to increase gene expression where a copy is placed under the control of a downstream promoter resulting in the expression of a hybrid protein [START_REF] Whoriskey | Genetic rearrangements and gene amplification in Escherichia coli: DNA sequences at the junctures of amplified gene fusions[END_REF] or the expression of the intact ORF under the control of another promoter [START_REF] Blount | Genomic analysis of a key innovation in an experimental Escherichia coli population[END_REF]. Classical models for gene duplication assume that there is no cost for the cell; newer models have shown that gene duplication has a substantial cost to cell fitness [START_REF] Reams | Duplication frequency in a population of Salmonella enterica rapidly approaches steady state with or without recombination[END_REF][START_REF] Adler | High Fitness Costs and Instability of Gene Duplications Reduce Rates of Evolution of New Genes by Duplication-Divergence Mechanisms[END_REF]. Therefore, due to the cost, tandem duplications tend to be unstable within a population and are often resolved by recombination before fixation [START_REF] Reams | Duplication frequency in a population of Salmonella enterica rapidly approaches steady state with or without recombination[END_REF]. To achieve fixation within a population, duplications need to achieve equilibrium between benefit and cost.

Limits to genome rearrangements

While the general consensus is that rearrangements occur randomly, there are several constraints that may affect the occurrence of DNA rearrangements.

First, selective forces preserve a certain amount of symmetry in the size of the two replichores of a circular chromosome between the origin and terminus of replication (Roth JR 1996;[START_REF] Eisen | Evidence for symmetric chromosomal inversions around the replication origin in bacteria[END_REF]. This may partly explain the strong conservation of gene order (synteny) between E. coli and Salmonella, although large DNA inversions can be observed in laboratory conditions where those selective pressures may be relaxed.

Second, the structural organisation of the E. coli chromosome has been shown to affect the probability of DNA rearrangements [START_REF] Esnault | Chromosome structuring limits genome plasticity in Escherichia coli[END_REF]. Specifically, the genome is physically organised into distinct macrodomains [START_REF] Boccard | Spatial arrangement and macrodomain organization of bacterial chromosomes[END_REF], and rearrangements affecting the replication origin or terminus domain and DNA inversions between the left and right macrodomains have been shown to be detrimental due to their effects on replication fork progression [START_REF] Esnault | Chromosome structuring limits genome plasticity in Escherichia coli[END_REF].

Rearrangements in Evolution Experiments

Chromosomal rearrangements have also been identified in evolution experiments whereupon bacterial strains are propagated for various times under controlled conditions. It can be theorised that this is due to the fact that many evolution experiments are a constant environment, therefore radical optimisation of the genome is highly beneficial in these circumstances. Deletions, duplications, large-scale inversions have all been detected in E. coli populations that have been propagated for various time lengths under different conditions. It is important to keep in mind that what is observed in these populations does not constitute the whole population as they are samples taken from each experiment. Two types of experiments are routinely used, batch cultures where populations routinely go through different growth phases and a dilution phase and continuous cultures where populations are in a constant environment without a dilution phase throughout the experiment. Batch cultures provide the largest amount of data as they have been more studies. During these experiments, there have been multiple deletions and amplification events that have conferred fitness advantages and even phenotypic innovation [START_REF] Schneider | Long-term experimental evolution in Escherichia coli. IX. Characterization of insertion sequence-mediated mutations and rearrangements[END_REF][START_REF] Cooper | Mechanisms causing rapid and parallel losses of ribose catabolism in evolving populations of Escherichia coli B[END_REF][START_REF] Blount | Genomic analysis of a key innovation in an experimental Escherichia coli population[END_REF]. In 3 out of 6 lines adapting to thermal stress there was evidence of duplications and deletions. 3 of the duplications affected the same chromosomal region containing 4 candidate genes for increased fitness in the conditions. The parallelism indicates that they were beneficial, even though it was not quantified [START_REF] Riehle | Genetic architecture of thermal adaptation in Escherichia coli[END_REF].

Continuous cultures of E.coli evolving in nutritional limitation chemostats have adapted to their environment including fixing a duplication containing the sigma factor rpoS. One of the copies then had a deletion of a part of the duplication. When clones were cultured outside its evolution environment the duplication was readily resolved to either the ancestral or the new (containing deletion) version (Fig 4). This shows that rearrangements can be specific to given environments and that rearrangements not previously seen can be observed in specific conditions. The exact beneficial advantages brought by this rearrangement are not known but could represent a second order process conferring a higher evolutionary potential [START_REF] Gaffe | Insertion sequence-driven evolution of Escherichia coli in chemostats[END_REF][START_REF] Maharjan | A case of adaptation through a mutation in a tandem duplication during experimental evolution in Escherichia coli[END_REF]. A recent study [START_REF] Sun | Genome-wide detection of spontaneous chromosomal rearrangements in bacteria[END_REF] based on whole genome sequencing of a bacterial population of Salmonella enterica var.

Typhimurium that was propagated for 240 generations in a chemostat revealed that duplications, inversions, and small deletions were detectable in ≥20% of the population after only 50 generations of growth.

III. Mobile genetic elements

Mobile genetic elements can be split into two groups, those able to transfer horizontally between chromosomes (plasmids and bacteriophages) and those that are only mobile between genetic material from within the cell or necessitate a vector for horizontal transfer (transposons and insertion sequences (IS)).

IS identification and transposase

Insertion sequences (IS) are among the smallest transposable element present in the prokaryote genome (between 700bp and 2,5kbp [START_REF] Mahillon | Insertion sequences[END_REF]. These elements are ubiquitous in almost every bacterial genome and all the information relevant to IS are stored in a specialised database called ISbiofinder [START_REF] Kichenaradja | ISbrowser: an extension of ISfinder for visualizing insertion sequences in prokaryotic genomes[END_REF].

A B

They are classified into families depending on (1) their primary sequence and their gene products [START_REF] Siguier | Bacterial insertion sequences: their genomic impact and diversity[END_REF] . Up to now over 2000 IS have been identified [START_REF] Mahillon | Insertion sequences[END_REF], organised into some 20 major families. Some of these IS contain additional functions encoded by partial translation of ORFs or on separate ORFs that regulate the levels of transposition and some contain genes of unknown function (for reviews see [START_REF] Nagy | Regulation of transposition in bacteria[END_REF][START_REF] Gueguen | Truncated forms of IS911 transposase downregulate transposition[END_REF] IS elements may act as simple transposable elements capable of replicating and/or translocating to different sites within the genome or onto plasmids in the bacterium, but IS elements are also responsible for the formation of composite transposons, by flanking on either side, unrelated genes causing the transposition of everything in between both IS as well as the IS [START_REF] Rosner | Transposition of IS1-lambdaBIO-IS1 from a bacteriophage lambda derivative carrying the IS1-cat-IS1 transposon (Tn9)[END_REF]. Upon insertion IS elements often create a direct duplication of the target site which varies depending on the element [START_REF] Mahillon | Insertion sequences[END_REF]. This can be up to 10bp creating a substantial effect even if the IS is later excised as the direct repeat is maintained in the genome. This is also variable for some IS elements [START_REF] Iida | Transposable element IS1 intrinsically generates target duplications of variable length[END_REF]. There are three main families of IS transposases, the classical and most abundant type being the DDE transposase (present in the major families IS1, IS3, Some IS elements, such as IS1 are capable of undergoing both types of transposition [START_REF] Biel | Mechanism of IS1 transposition in E. coli: choice between simple insertion and cointegration[END_REF]. IS1 undergoes replicative transposition in 3/4 cases and conservative transposition is due to an imperfect replication.

Effect of IS transpositions on gene expression

IS elements have strong effects on the regions that they inserted into. The most common effect is gene inactivation due to the IS disrupting the coding sequence [START_REF] Kumar | IS30-related transposon mediated insertional inactivation of bile salt hydrolase (bsh1) gene of Lactobacillus plantarum strain Lp20[END_REF]). This is one of the most efficient ways the genome has of disrupting potentially superfluous gene expression [START_REF] Nakamura | Inactivation of the Serratia marcescens gene for the lipoprotein in Escherichia coli by insertion sequences, IS1 and IS5; sequence analysis of junction points[END_REF][START_REF] Park | Enhancing recombinant protein production with an Escherichia coli host strain lacking insertion sequences[END_REF]). Due to highly mobile nature of IS elements, this gene inactivation is potentially reversible if the direct repeat left by the IS element is not disruptive. This is of particular concern in pathogens as IS elements play a regular role in the activation of pathogenic genes which can be selected for in the right environments [START_REF] Ziebuhr | A novel mechanism of phase variation of virulence in Staphylococcus epidermidis: evidence for control of the polysaccharide intercellular adhesin synthesis by alternating insertion and excision of the insertion sequence element IS256[END_REF]. This can lead to activation or increased expression of antibacterial resistance genes especially for multidrug efflux systems, creating a major health problem (Jellen-Ritter and Kern 2001; [START_REF] Aubert | Functional characterization of IS1999, an IS4 family element involved in mobilization and expression of beta-lactam resistance genes[END_REF]. Gene expression can also be modified by insertion of IS elements into promoter regions. This can either reduce expression but can also increase expression levels if the IS is inserted into a repressor sequences. IS can also have an impact on gene expression because several have outward facing promoter regions (for example -35 boxes [START_REF] Prentki | Functional promoters created by the insertion of transposable element IS1[END_REF]Flechard 2014), when inserted in phase and in the correct context with a gene they can activate its transcription [START_REF] Camarena | An IS4 insertion at the glnA control region of Escherichia coli creates a new promoter by providing the -35 region of its 3'-end[END_REF][START_REF] Kallastu | Identification and characterization of IS1411, a new insertion sequence which causes transcriptional activation of the phenol degradation genes in Pseudomonas putida[END_REF].

Insertion specificity of IS elements

Most of the IS target sites are thought to be randomly distributed, however some IS elements have a preference for certain sequence characteristics, potentially biasing the genes that are affected by IS transposition. For example the transposase of IS903 is able to form dimers or multimers and recognise specific sequences inducing transposition to specific areas [START_REF] Hu | Target choice and orientation preference of the insertion sequence IS903[END_REF]. The more common IS1 has a preference for AT rich sequences through interaction with the transposase [START_REF] Zerbib | Specificity of insertion of IS1[END_REF]. Some IS show a preference for certain DNA secondary structures such as repeated extragenic palindromes [START_REF] Clement | IS1397 is active for transposition into the chromosome of Escherichia coli K-12 and inserts specifically into palindromic units of bacterial interspersed mosaic elements[END_REF][START_REF] Tobes | Bacterial repetitive extragenic palindromic sequences are DNA targets for Insertion Sequence elements[END_REF], integrons [START_REF] Tetu | A family of insertion sequences that impacts integrons by specific targeting of gene cassette recombination sites, the IS1111-attC Group[END_REF] or other even IS elements [START_REF] Hallet | IS231A from Bacillus thuringiensis is functional in Escherichia coli: transposition and insertion specificity[END_REF].

Control of IS transposition

The transposition of IS elements is strictly controlled by a multitude of mechanisms and is generally kept at low levels. This is probably to offset the deleterious effects of excessive transposition as most mutations are associated with negative effects on bacteria [START_REF] Doolittle | Selfish DNAs with self-restraint[END_REF]. IS elements auto regulate their transposition activity in several ways that can be complementary, these are the most common mechanisms with several other mechanisms are specific to a small number of IS elements. I) The most common IS families (families IS1, IS3, IS4 and IS5) have an ORF that codes for small transcriptional repressors of the transposase as either a separate ORF or more commonly as a truncated version of the transposase [START_REF] Zerbib | The regulatory role of the IS1-encoded InsA protein in transposition[END_REF][START_REF] Escoubas | Translational control of transposition activity of the bacterial insertion sequence IS1[END_REF]. II) Several IS families (families IS1, IS3, IS5 and IS630) require a programmed ribosomal frameshift during translation of the transposase RNA [START_REF] Escoubas | Translational control of transposition activity of the bacterial insertion sequence IS1[END_REF][START_REF] Vogele | High-level ribosomal frameshifting directs the synthesis of IS150 gene products[END_REF]. This commonly leads to 3 gene products being expressed, InsAB -the transposase, insA-a regulatory protein and insB of unknown function [START_REF] Vogele | High-level ribosomal frameshifting directs the synthesis of IS150 gene products[END_REF]Haas and Rak 2002). III) transposase is unstable. The transposase of IS903 and IS50 are sensitive to degradation by the Lon protease [START_REF] Derbyshire | Role of instability in the cis action of the insertion sequence IS903 transposase[END_REF][START_REF] Koonin | Computer-assisted dissection of rolling circle DNA replication[END_REF] and the transposase of IS911 is sensitive to exposure to 42°C [START_REF] Haren | IS911-mediated intramolecular transposition is naturally temperature sensitive[END_REF].

IV) many IS elements, through the formation of secondary structures in mRNA initiated from outside the IS, protect themselves from impinging transcription [START_REF] Davis | Tn10 protects itself at two levels from fortuitous activation by external promoters[END_REF][START_REF] Krebs | Transcriptional and translational initiation sites of IS50. Control of transposase and inhibitor expression[END_REF].V) small residual IS elements are capable of producing truncated transposases that bind with functional transposases to regulate transposition [START_REF] Gueguen | Truncated forms of IS911 transposase downregulate transposition[END_REF]. VI) certain IS elements lack transcriptional stop sites and relay on insertion next to stop codons to have a functioning transcription of the transposase [START_REF] De Meirsman | Nucleotide sequence analysis of IS427 and its target sites in Agrobacterium tumefaciens T37[END_REF]). VII) Antisense RNA regulates the translation of IS10 transposase and impairs the binding of the ribosome to the RBS [START_REF] Jain | Models for pairing of IS10 encoded antisense RNAs in vivo[END_REF].

IS elements generally exist in low copy numbers in the bacterial genome and their transposition is highly limited [START_REF] Mahillon | Insertion sequences[END_REF]. It is hypothesised that they are a recent addition the bacterial genome as they are highly conserved between species on the other hand their functionality stems from their highly conserved sequence. The origin and capture of IS elements in prokaryotes is not well understood and none have been catalogued.

However, DDE transposases, specifically from the IS3 family, are closely related to the retroviral integrase catalytic core (IN) which could indicate an evolutionary link [START_REF] Haren | Integrating DNA: transposases and retroviral integrases[END_REF]. The persistence of IS elements in the genome is a topic of debate and there emerges two hypotheses. The first hypothesis is that movements of IS elements have mostly negative effects on genomes by breaking genes and their functions [START_REF] Charlesworth | The evolutionary dynamics of repetitive DNA in eukaryotes[END_REF]. Therefore they are maintained in the cell by a high level of replication to additional genomic sites and onto plasmids, this coupled with horizontal transfer allows for the persistence of an element even if it's deleterious to the cell. The second hypothesis is that even though the majority of IS related events are deleterious they are capable of creating enough beneficial mutations, through insertion, to allow for their fixation within their host (Schneider and Lenski 2004).

Evidences exist for the beneficial effects of IS elements. IS elements are capable of rapid expansion in copies numbers present on a genome. This leads to massive amounts of gene inactivation, genome rearrangements and genome reduction which in certain contexts is a beneficial adaptation. While the exact nature of control over this phenomena is not known, there is a correlation with high levels of IS elements and the recent passage of a bacteria to a facultative endosymbiont [START_REF] Bordenstein | Mobile DNA in obligate intracellular bacteria[END_REF]Touchon and Rocha 2007;[START_REF] Gil | Massive presence of insertion sequences in the genome of SOPE, the primary endosymbiont of the rice weevil Sitophilus oryzae[END_REF]). One explanation could be that generally IS movement is negatively selected for, as it has strong polar effects, until the host finds itself in an stable environment where it rends genes redundant due to non-expression or even constitutive expression that is not beneficial (i.e. the passage to facultative endosymbiont) and therefore IS movements won't have such large effects as there are more targets with neutral effects and they will not be counter selected (Touchon and Rocha 2007). The inactivation of genes would even represent a fitness gain for the host in these circumstances, so much so that IS elements can account for up to 23% of the genome of newly formed endosymbionts [START_REF] Gil | Massive presence of insertion sequences in the genome of SOPE, the primary endosymbiont of the rice weevil Sitophilus oryzae[END_REF][START_REF] Schmitz-Esser | A bacterial genome in transition--an exceptional enrichment of IS elements but lack of evidence for recent transposition in the symbiont Amoebophilus asiaticus[END_REF]. Several different IS elements are able to sweep through populations in these case creating a genome that has a high level of homologous sequences [START_REF] Cerveau | Short-and long-term evolutionary dynamics of bacterial insertion sequences: insights from Wolbachia endosymbionts[END_REF]. This represents the step before the massive genome reduction that is more commonly associated with endosymbionts. However, the exact mechanisms for IS increase is not understood, while there is evidence that higher transposition rates increased levels of IS is thought to be more of a stochastic process [START_REF] Kleiner | Metaproteomics reveals abundant transposase expression in mutualistic endosymbionts[END_REF]. This process could also apply to any evolving population that is kept in a steady environment over a long period of time. E.coli was cultured for up to 480 generations in a glucose limited chemostat. This represents a stable environment where there can clearly be redundancy in the genome as genes are not expressed. As such, IS elements largely participated in the adaptation of several populations. Changes included insertion of IS1 into the stationary phase sigma factor rpoS and insertion of IS5 into a gene controlling mutation rates [START_REF] Gaffe | Insertion sequence-driven evolution of Escherichia coli in chemostats[END_REF]. Significant IS movements were also categorised in other evolution experiments. IS10 is one of the major drivers in the evolution of E.coli to osmotic pressure with a beneficial mutation linked to IS10 present in all ten evolved populations (Stoebel and Dorman 2010).

With the increase in IS elements over the whole genome there is an increase in homologous targets. This allows for virtually any rearrangement to potentially occur. For example in the, new insertions of IS150 create the opportunity for the deletion of ribose operon in evolving populations [START_REF] Cooper | Mechanisms causing rapid and parallel losses of ribose catabolism in evolving populations of Escherichia coli B[END_REF]. IS150 is equally important for evolution to freeze thaw cycles in E.coli [START_REF] Sleight | Genetic basis of evolutionary adaptation by Escherichia coli to stressful cycles of freezing, thawing and growth[END_REF]. IS elements are also important for the adaptation of Lactococcus lactis to cycles of growth/starvation [START_REF] Sleight | Genetic basis of evolutionary adaptation by Escherichia coli to stressful cycles of freezing, thawing and growth[END_REF]) and to allow the utilisation of cobalt in Methylobacterium extorquens [START_REF] Chou | Fast growth increases the selective advantage of a mutation arising recurrently during evolution under metal limitation[END_REF].

It is therefore of crucial importance to understand the evolutionary role of IS elements and in particular their relative involvement in the overall bacterial mutagenesis. There have been two methods to approach this question until now. The first is experimental measures of IS transposition with tools that are linked to particular genetic loci. In one experiment the insertion of IS was determined in the LacZ loci, this is an especially interesting tool to quantify IS movement rates [START_REF] Huisman | A new generalizable test for detection of mutations affecting Tn10 transposition[END_REF]. Another approach uses a system where a reporter gene lacking transcription and translational start points is flanked by IS1

IRs. The plasmid also contains the IS1 transposase without the ribosomal frameshift which forces an under regulation of transposase and a resulting increase in transposition. The GFP is only produced if it transposes in the correct location. This gives an estimate for IS1 transposition [START_REF] Saito | Measurements of transposition frequency of insertion sequence IS1 by GFP hop-on assay[END_REF]). However, these approaches have its drawbacks as it doesn't give a global view of IS movements in the genome as some IS have specific hotspots which could be located outside the target loci and there are different IS present on the genome. The second drawback is that this approach does not quantify the link between natural selection and IS activity as the bacteria are not under a well-defined selective pressure. The second method used to quantify the involvement of IS in bacterial mutagenesis involves looking at the distribution of IS elements in closely related bacteria through RFLP or genome sequencing. This approach also has various limitations as it doesn't allow the analysis of the dynamics of IS movements throughout its evolution (i.e did IS distribution occur as a burst or as a stochastic method?) as all that is analysed is essentially bacteria that have diverged a long (evolutionary) time ago. To overcome this one evolution experiment involving 50 replicates of E.coli were analysed for IS movements over 1610 generations IS transposition rates were estimated at 1.15 × 10 -5 per element per generation, unfortunately this approach is limited to a small timescale and doesn't have the resolution over a longer period of time [START_REF] Sousa | Rates of transposition in Escherichia coli[END_REF].

IV. The Long Term Evolution Experiment (LTEE)

Figure 7: Method for the Long Term Evolution Experiment (LTEE). Twelve independent cultures founded from a common ancestor have been propagated by daily serial transfer in glucose limited minimal media (Davis media). Over 60,000 generations have been obtained at this day and regular samples have been taken every 500 -1000 generations and conserved at -80°C.

Experiment set-up

The longest ongoing evolution experiment was initiated by Richard Lenski in 1988. Twelve populations were founded from an E.coli B strain acting as a common ancestor [START_REF] Lenski | The E. coli long-term experimental evolution project site[END_REF]. They are daily propagated after a 1/100 dilution in 10 ml Davies Minimal media supplemented with 25mg/L of glucose creating an environment where cells alternate between glucose abundance to glucose starvation and are selected through a bottleneck every 24 hours. There is roughly 6.64 generations per 24 hour cycle with the final cell count arriving at ~5x10 7 cells per ml (Lenski 1991) from 10 5 cells per ml with the ancestor. Arabinose is used as a marker for competition experiments and is shown to be neutral under the evolution experiment conditions (Lenski 1991). Every 500 generations a sample is taken from each population and is frozen. The frozen sample can be later revived and therefore constitutes a frozen fossil record of the evolving populations, allowing for comparison between the ancestor and evolved clones. The ancestor is not naturally competent and is asexual; therefore any evolution that occurs is due to genetic drift and natural selection (Lenski 1991;[START_REF] Lenski | Phenotypic and Genomic Evolution during a 20,000-Generation Experiment with the Bacterium Escherichia coli[END_REF]). The populations were named Ara+1 to Ara+6 and Ara-1 to Ara-6, alluding to their capacity to utilise arabinose as a carbon source for growth. The populations have now been evolving for over 60,000 generations in these conditions

Common evolution between the populations

The twelve populations rapidly adapted to their environments, showing an increase in fitness compared to their ancestor (Cooper and Lenski 2000;[START_REF] Wiser | Long-term dynamics of adaptation in asexual populations[END_REF]. The fitness increased by almost 50% in the first 5000 generations and then the increase was less marked, increasing by around 25% more over the following 15,000 generations (Cooper and Lenski 2000;Philippe, Crozat et al. 2007) (Fig 8). Not only did they increase in fitness in relations to the media, they also underwent many other phenotypic changes. They all had an increase in cell size, different cell morphology, a decrease in lag phase and an increase in maximal growth rates [START_REF] Lenski | Dynamics of adaptation and diversification: a 10,000-generation experiment with bacterial populations[END_REF][START_REF] Lenski | Phenotypic and Genomic Evolution during a 20,000-Generation Experiment with the Bacterium Escherichia coli[END_REF][START_REF] Philippe | Evolution of global regulatory networks during a longterm experiment with Escherichia coli[END_REF][START_REF] Philippe | Evolution of penicillin-binding protein 2 concentration and cell shape during a long-term experiment with Escherichia coli[END_REF]. They all presented an increase in their capacity to utilise glucose and lost the capability to grow on certain carbon sources, most notably, all twelve populations had a deletion in the ribose operon [START_REF] Cooper | Mechanisms causing rapid and parallel losses of ribose catabolism in evolving populations of Escherichia coli B[END_REF]) and they all presented deficiencies in the operon for the use of maltose [START_REF] Pelosi | Parallel changes in global protein profiles during longterm experimental evolution in Escherichia coli[END_REF].

Genetic analysis of the twelve populations.

The twelve populations show large changes in the regulation networks [START_REF] Hindre | New insights into bacterial adaptation through in vivo and in silico experimental evolution[END_REF]. The rewiring of the global regulatory networks was mainly due to mutations in spoT in all twelve populations [START_REF] Cooper | Parallel changes in gene expression after 20,000 generations of evolution in Escherichiacoli[END_REF]. This was not the only mutation common to many populations. Mutations in fis, dusB and topA were found to increase the levels of supercoiling of the DNA of the majority of the twelve populations [START_REF] Crozat | Long-term experimental evolution in Escherichia coli. XII. DNA topology as a key target of selection[END_REF][START_REF] Crozat | Parallel genetic and phenotypic evolution of DNA superhelicity in experimental populations of Escherichia coli[END_REF]. Two other genes, pykF and nadR were found to be mutated in every population. It is noteworthy that the mutations were not identical and could affect different parts of the gene [START_REF] Woods | Tests of parallel molecular evolution in a long-term experiment with Escherichia coli[END_REF]). This high level of parallelism for these genes between the populations indicates that they are part of the evolution and adaptation to the evolution experiment conditions. This is confirmed when the mutations were reconstructed in ancestral genetic backgrounds, conferring to them an advantage. Analysis of early mutations in one population shows that there is a high level of negative epistasis between positive mutations with the more beneficial the mutation, the stronger the effect of negative epistasis [START_REF] Khan | Negative epistasis between beneficial mutations in an evolving bacterial population[END_REF]. The only parallel rearrangement that has been shown to be beneficial is the deletion of the ribose operon in all twelve populations. The deletion occurred between an ancestral IS150 copy and a new IS150 copy that inserted into the operon downstream. In all twelve populations the new IS150 copy inserted into different positions but they were all within the operon [START_REF] Cooper | Mechanisms causing rapid and parallel losses of ribose catabolism in evolving populations of Escherichia coli B[END_REF]). However there are many more evolved alleles that are common in several populations that have either no detectable or even slightly deleterious effects on fitness [START_REF] Crozat | Parallel genetic and phenotypic evolution of DNA superhelicity in experimental populations of Escherichia coli[END_REF].

Diversity between the populations.

Even though there is a high level of parallelism between the populations each population has evolved different characteristics. Strikingly, there are 6 populations that have evolved higher mutation rates than the ancestor through mutations in their DNA repair mechanisms [START_REF] Sniegowski | Evolution of high mutation rates in experimental populations of E. coli[END_REF][START_REF] Wielgoss | Mutation rate dynamics in a bacterial population reflect tension between adaptation and genetic load[END_REF]. In the population Ara-1 a mutations in mutT which increase mutation rates by~150 fold [START_REF] Wielgoss | Mutation rate dynamics in a bacterial population reflect tension between adaptation and genetic load[END_REF] before a subsequent mutation in mutY that reduces the mutation rate by ~40-60% [START_REF] Wielgoss | Mutation rate dynamics in a bacterial population reflect tension between adaptation and genetic load[END_REF]. High levels of IS movements have also been observed for one population (Ara+1) in particular, mimicking higher SNP rates [START_REF] Papadopoulos | Genomic evolution during a 10,000generation experiment with bacteria[END_REF][START_REF] Schneider | Long-term experimental evolution in Escherichia coli. IX. Characterization of insertion sequence-mediated mutations and rearrangements[END_REF]. Several populations were also analysed for large chromosomal rearrangements at the same time as being analysed for IS elements [START_REF] Schneider | Long-term experimental evolution in Escherichia coli. IX. Characterization of insertion sequence-mediated mutations and rearrangements[END_REF]. At 2,000 generations one clone in the population Ara+1 had an inversion of over 1/3 of its genome. This clone is then established in the population.

Interestingly, this is the population where there is most movement of IS elements. In Ara-1, analysis of the distribution of IS elements and whole-genome sequencing detected four large deletions with sizes ranging from ~8 to ~23 kbp and an inversion of one-third of the chromosome that is different from the Ara+1 inversion [START_REF] Schneider | Long-term experimental evolution in Escherichia coli. IX. Characterization of insertion sequence-mediated mutations and rearrangements[END_REF]Barrick, Yu et al. 2009) (Fig 9). The effect of rearrangements in the populations is difficult to estimate as there are no isogenic clones without the rearrangement and to reconstruct any rearrangement would necessitate complex genomic manipulation with a strong possibility of introducing new mutations. At the most it is possible to estimate that a given rearrangement is beneficial through parallelism [START_REF] Woods | Tests of parallel molecular evolution in a long-term experiment with Escherichia coli[END_REF]). So while the populations have been subject to parallel evolution, certain populations have taken unique evolutionary trajectories. This is especially true for two populations, Ara-2 and Ara-3.

Evolution of polymorphism in Ara-2

The population Ara-2 evolved along a unique evolutionary path. The population split in to two ecotypes called S and L, one (S) characterised by small colony formers and small cells,

A B
the other (L) by large colonies and large cell sizes [START_REF] Rozen | Long-Term Experimental Evolution in Escherichia coli. VIII. Dynamics of a Balanced Polymorphism[END_REF][START_REF] Rozen | Long-term experimental evolution in Escherichia coli. XIII. Phylogenetic history of a balanced polymorphism[END_REF]. This was unexpected because the experiment was designed to minimise ecological niches by firstly limiting metabolite production by glucose utilisation (principally acetate) and secondly by limitation of cell numbers. First detected at 6500 generations, the clones co-exist in a dynamic relationship for over 50,000 generations and are still present today [START_REF] Rozen | Long-Term Experimental Evolution in Escherichia coli. VIII. Dynamics of a Balanced Polymorphism[END_REF][START_REF] Gac | Ecological and evolutionary dynamics of coexisting lineages during a long-term experiment with Escherichia coli[END_REF][START_REF] Plucain | Epistasis and allele specificity in the emergence of a stable polymorphism in Escherichia coli[END_REF]. Not only were they present in co-existence, based on IS profiles, there was no genetic exchange between the two populations, which based on classical markers indicates that they two separate populations [START_REF] Rozen | Long-term experimental evolution in Escherichia coli. XIII. Phylogenetic history of a balanced polymorphism[END_REF]. The emergence of the populations can be traced back to essentially 3 mutations. Before the divergence the gene spoT is mutated and is found in both sub-populations. Mutations in arcA and gntR then occurred in the S lineage but are not present in the L. These three mutations together are responsible for S phenotypes and

Evolution of a new catabolic pathway Ara-3

The population Ara-3 presents a very special case of evolution. Historically the ancestor is not capable of internalising citrate and therefore using it as a carbon source for growth [START_REF] Koser | Correlation of Citrate Utilization by Members of the Colon-Aerogenes Group with Other Differential Characteristics and with Habitat[END_REF]. However in the evolution media there is citrate present along with the glucose, the principal carbon source. There exist several cases where citrate utilisation (cit+) in E. coli is acquired through either horizontal transfer of genes or several mutations to a residual citrate transport system [START_REF] Ishiguro | Plasmids in Escherichia coli controlling citrate-utilizing ability[END_REF][START_REF] Hall | Chromosomal mutation for citrate utilization by Escherichia coli K-12[END_REF]. At around 33,000 generations clones from the population Ara-3 had evolved to utilise the citrate as a carbon source [START_REF] Blount | Historical contingency and the evolution of a key innovation in an experimental population of Escherichia coli[END_REF]. This has an important fitness increase for the clone as it is able to achieve higher cell numbers in the population. The appearance of this trait is highly dependent on the genetic background of the clone. When evolution was replayed, clones from later generations were more likely to produce cit+ progeny than the ancestor, earlier clones or contemporary clones sampled from other populations. In effect, the genetic backgrounds of the evolved clones potentiated the clone for the evolution of citrate utilisation. When genome sequences were available for the population, the genetic history was exposed (Fig 11). There were in fact 3 potentiated clades that coexisted over 10,000 generations that were capable in producing cit+ progeny in replay experiments. The clade that is capable of using citrate had a tandem duplication event of the gene citT, the principal gene for citrate transport (Blount, Z. D., J. E. Barrick, et al. 2012). The duplication event placed the gene behind an aerobically expressed promoter (Fig 12). This allowed for the transport protein to be expressed in the experimental conditions. The first clone sequenced with the duplication is in fact at 31,500 generations and has a poor utilisation of citrate that would represent an actualisation step. Further modifications to regulatory networks explain the increased use of citrate. To confirm the potentiated nature of the clone the evolution experiment was replayed and citrate+ clones were analysed to see if by what mechanism they had evolved to utilise citrate. Out of 19 reevolved clones only 8 had new duplications. They had the same effect as the first duplication, placing the citrate transport genes behind a strong promoter. In six clones an IS3 inserted just before the gene. IS3 has an outward facing promoter sequence and was thus able to jump start gene expression [START_REF] Charlier | IS3 can function as a mobile promoter in E. coli[END_REF]. Two mutants have large duplications encompassing all or part of the cit operon. One mutant has a large inversion that places most of that operon downstream of the promoter for the fimbria regulatory gene fimB, and another has a deletion in citG that presumably formed a new promoter. Also, most of these mutants have stronger phenotypes than the earliest Cit + clones in the main experiment. Evolved genome (adapted from Blount, Z. D., J. E. Barrick, et al. 2012).

As such we know of only very little of the rearrangements present during the evolution experiment. We know of only examples that have had a dramatic consequence in the fitness of populations, the deletion of the ribose operon and the duplication of citT. The only general study was done on the populations Ara+1 and Ara-1 up to 20,000 generations. Even though there are a large number of sequences available at different time points for the twelve populations, chromosomal rearrangements such as inversion and duplications could be missed sue to sequencing nature. In this study I will identify large chromosomal rearrangements that have evolved and also characterise a duplication present in several populations. The second part of my thesis I will investigate the role of IS elements in the populations, with particular focus on the population Ara+1 which presented an unusually high number of IS elements at 20,000 generations. Together these two aspects will help further characterise the populations in understudied areas.

Large Chromosomal Rearrangements during a Long-Term Evolution Experiment with

Escherichia coli

Colin Raeside, a,b Joël Gaffé, a,b Daniel E. Deatherage, c Olivier Tenaillon, d,e Adam M. Briska, f † Ryan N. Ptashkin, f ‡ Stéphane Cruveiller, g,h Claudine Médigue, g,h Richard E. Lenski, i,j Jeffrey E. Barrick, c,j Dominique Schneider having between 5 and 20 such events. In three populations, successive rearrangements affected particular genomic regions. In five populations, the rearrangements affected over a third of the chromosome. Most rearrangements (~70%) involved recombination between IS elements, illustrating their importance in mediating genome plasticity. Two lines of evidence suggest that many of these rearrangements may confer higher fitness. First, parallel changes were observed across the independently evolving populations, with ~65% of the rearrangements affecting the same loci in at least two populations. For example, the riboseutilization operon and the manB-cpsG region were deleted in 12 and 10 populations, respectively, suggesting positive selection, and this inference was previously confirmed for the former case. Second, optical maps from additional clones sampled over time from one population showed that most rearrangements occurred early in the experiment, when fitness was increasing most rapidly. Therefore, large-scale genomic rearrangements evidently affected evolutionary outcomes in these populations.

IMPORTANCE Bacterial chromosomes are dynamic structures shaped by long histories of evolution. Among genomic changes, large-scale DNA rearrangements can have large effects on the presence, order, and expression of genes. Whole-genome sequencing that relies on

Introduction

Large-scale chromosomal rearrangements have played important roles in long-term organismal evolution (1,2), including in processes of speciation (3,4) and genome reduction (5). On shorter time scales, even single rearrangement events such as duplications, amplifications, inversions, deletions and translocations can have profound effects on organismal phenotypes, typically by altering gene regulation or disrupting genes. In bacteria, some genome rearrangements have led to traits important for virulence (6), and rearrangements are sometimes even developmentally regulated (7).

Large-scale rearrangements have been identified in diverse bacteria-Gram-negative, Gram-positive, pathogenic, and non-pathogenic-including Escherichia coli (8), Salmonella typhi, Yersinia pestis, Helicobacter pylori, Mycobacterium leprae (9,10), Pseudomonas stutzeri (11), Pseudomonas aeruginosa (12), Francisella tularensis (13), Neisseria gonorrhoeae (14), Lactococcus lactis (15), and Staphylococcus aureus (16). Besides their effects on chromosome structure, bacterial DNA rearrangements may cause phenotypic changes by the incorporation of foreign DNA into host genomes through horizontal gene transfer (17,18), by changes in gene expression (19), or by genome reduction through the loss of non-essential genes (20). The major mechanisms producing chromosomal rearrangements are recombinational exchanges between homologous sequences including ribosomal operons (21) as well as mobile genetic elements such as transposons ( 16), insertion sequence (IS) elements (15), and prophages (8). Comparisons of related genomes often reveal numerous DNA inversions among other rearrangements (9,22,23).

Some constraints may influence the occurrence of DNA rearrangements. First, selection may preserve symmetry in the size of the two replichores of a circular chromosome between the origin and terminus of replication (9,24). This selection may help explain the strong conservation of gene order (synteny) between E. coli and Salmonella, although large inversions have been observed under laboratory conditions where such selection may be relaxed. Second, the structural organization of the E. coli chromosome can affect rearrangements (25). Specifically, the genome is organized into distinct macrodomains (26), and rearrangements affecting the replication origin or terminus domain and inversions between the left and right macrodomains have been shown to be detrimental owing to their effects on replication-fork progression (25).

Large-scale rearrangements occur spontaneously at measurable frequencies, although the rates at which they occur are uncertain. In an older study, duplications were reported to arise at frequencies between 10 -2 and 10 -5 per cell division, depending on their chromosomal location (21). A more recent whole-genome sequencing study (27) of a population of Salmonella enterica var. Typhimurium that was propagated in a chemostat found duplications, inversions, and small deletions in ≥20% of the cells after only 50 generations.

There are several possible explanations for these differences. First, it is generally difficult to disentangle underlying mutation rates from the effects of selection that may cause some mutants to replicate faster or slower than non-mutant cells. Second, the earlier study involved plating to isolate clonal genotypes, which may induce the loss of unstable rearrangements including duplications, thereby underestimating their true frequency (28). Third, earlier studies were based on observable phenotypes that were produced after the rearrangements occurred and might have missed many other events (29)(30)(31)(32).

Chromosomal rearrangements have been discovered in many evolution experiments in which bacterial populations are propagated under various laboratory conditions. Deletions, duplications, and large-scale inversions have been detected in E. coli populations propagated in batch (33)(34)(35) and chemostat (28,36) cultures as well as under stressful conditions (37).

Rearrangements have also been found to occur in P. aeruginosa populations evolving in cystic fibrosis patients (38). In some cases, specific rearrangements have been shown to confer increased fitness or phenotypic innovations in these evolution studies (28,34,35,37).

To date, however, no study has attempted to provide an exhaustive analysis of the multiple, large-scale chromosomal rearrangements that have arisen during a long evolution experiment.

Rearrangements have usually been investigated using methods such as pulsed-field gel electrophoresis, microarray-based hybridization experiments, and whole-genome sequencing. Despite the name, even "whole-genome sequencing" typically relies on sequencing short DNA fragment libraries and therefore cannot detect certain large-scale rearrangements including inversions and other events involving long sequence repeats. To complement sequencing data, Whole Genome Mapping (hereafter, optical mapping)

techniques have been developed that produce a high-resolution, physically ordered restriction map of bacterial genomes (39)[START_REF] Shukla | Optical mapping reveals a large genetic inversion between two methicillin-resistant Staphylococcus aureus strains[END_REF][START_REF] Turner | Optical mapping and sequencing of the Escherichia coli KO11 genome reveal extensive chromosomal rearrangements, and multiple tandem copies of the Zymomonas mobilis pdc and adhB genes[END_REF]. We produced optical chromosomal maps for clones isolated from a long-term evolution experiment (LTEE) with E. coli. In this on-going experiment, 12 populations have been independently propagated from a common ancestor in the same glucose-limited minimal medium for more than 25 years and 50,000 cell generations. These evolving populations have adapted to the experimental environment and have increased in competitive fitness relative to the ancestor by more than 70%, on average [START_REF] Wiser | Long-term dynamics of adaptation in asexual populations[END_REF].

Some chromosomal rearrangements have been previously detected in the LTEE populations by using other techniques. However, with the exception of deletions involving the ribose operon found in all 12 populations (34), few rearrangements have been analyzed in detail. The ribose deletions were shown to occur at a high rate as well as to confer a slight fitness benefit in the LTEE conditions. In addition, other rearrangements have been detected

in three populations (designated Ara+1, Ara-1 and Ara-3). In Ara+1, an analysis of the distribution of IS elements by Southern blotting revealed an inversion of about one-third of the chromosome by recombination between two copies of IS150 (33). A substantial increase in the IS150 copy number also occurred in this population [START_REF] Papadopoulos | Genomic evolution during a 10,000-generation experiment with bacteria[END_REF]. In Ara-1, analysis of the distribution of IS elements and whole-genome sequencing found four large deletions (ranging from ~8 to ~23 kbp) and an inversion of one-third of the chromosome that is different from the inversion in Ara+1 (33,[START_REF] Barrick | Genome evolution and adaptation in a long-term experiment with Escherichia coli[END_REF]. The fitness consequences of these rearrangements are unknown. In Ara-3, numerous deletions, duplications and amplifications were detected in evolved clones by using genome sequencing data, including a specific tandem duplication and further amplification events involved in the production of a novel Cit + phenotype (35).

In an effort to obtain a more complete picture of the number and types of rearrangements that were substituted over time in all 12 LTEE populations, we combined optical mapping, genome sequencing, and targeted PCR and Sanger sequencing to analyze a total of 19 clones including a single clone sampled at 40,000 generations from each population as well as additional clones sampled at each of 7 other time points from population Ara-1. The resolution of optical maps cannot reliably detect rearrangements smaller than ~5 kbp (including, for example, new insertions of IS elements) unless they alter restriction sites. Nevertheless, we found that all 12 populations experienced large-scale chromosomal rearrangements and that most of these involved IS elements or other repeated sequences. Moreover, we saw many cases of parallel evolution across the populations in the genes that were affected by these rearrangements. Also, three populations had undergone complex rearrangements that involved successive inversion events.

Results

Chromosomal rearrangements in the twelve populations of the long-term evolution experiment. We combined optical mapping and genome sequence analyses to identify the precise location and borders of all large-scale chromosomal rearrangements that occurred in one clone sampled at 40,000 generations from each of the twelve E. coli populations of the LTEE (Fig. 1, Table S1). Combining these approaches allowed us to resolve rearrangements between large repeated elements, which is difficult or impossible with genome sequencing data alone, and to map the borders of the rearrangements with single-nucleotide resolution, which is impossible with optical mapping data alone. We also verified the rearrangement borders for two evolved clones from populations Ara+1 and Ara+2 in which we detected more complex rearrangements using PCR and Sanger sequencing. Primer pairs were designed adjacent to repeat sequences including IS elements and rRNA-encoding genes for these assays. The results agreed with our predictions in all cases, giving us confidence that the events we inferred in other clones are also accurate. Note, however, that optical mapping cannot detect IS insertion events because most rearrangements smaller than ~5 kbp are too small to resolve unless they affect restriction sites.

We identified a total of 110 rearrangement events in the twelve 40,000-generation clones, including 82 deletions, 19 inversions, and 9 duplications (Fig. 1, Table S1). Among the inversions, nine were involved in successive series of events that occurred over time in three populations (see next section). Among the duplications, three apparently involved successive events in which a typical tandem duplication was followed by deletion of the junction between the two duplicated copies, thereby resulting in an imperfect duplication (see next section).

Large deletions were the most frequent type of rearrangement, and they were found in all twelve populations, ranging in size up to ~55 kbp. Prophage remnants were often affected by these deletions; 30 of the 82 large deletions (36.6%) resulted in the loss of prophage DNA, although these regions cover only ~4% of the ancestral genome. This over-representation of prophage DNA is highly significant (binomial test P < 2.2e-16). The 19 inversions were found in nine populations, ranging in size from ~164 kbp to ~1.8 Mbp (see Fig. 1 legend for explanation of inversion sizes). In seven cases (one each in populations Ara+1, Ara-1, and Ara-5, and two each in Ara+2 and Ara-3), more than a quarter of the chromosome was affected by the inversions (Fig. 1). Successive inversions were inferred in some cases, and they were confirmed by examining multiple evolved clones from different generations (see next section). Nine duplications, ranging in size from ~3 kbp to ~180 kbp, were found in clones from four populations, including three with further deletions of the copy junctions (see next section).

The total number of rearrangements after 40,000 generations ranged from 5 in population Ara+3 to 20 in Ara-3, with an average of 9 rearrangements per population (Fig. S1). By that time, six of the 12 populations (Ara+3, Ara+6, Ara-1, Ara-2, Ara-3, and Ara-4)

had evolved hypermutable phenotypes that caused greatly elevated point mutation rates compared to the ancestor [START_REF] Wiser | Long-term dynamics of adaptation in asexual populations[END_REF]. We did not characterize the chronology of the rearrangements in relationship to the hypermutator status of each population, but there is no suggestion of any difference in the overall rate at which rearrangements accumulated in mutator versus nonmutator populations (Poisson test P = 1).

Most rearrangements occurred by recombination between repeated sequences, including 76 between homologous IS copies, 7 between the manB and cpsG genes (which share 96% sequence identity), and 5 between rRNA-encoding operons (Table S1, Fig. S1).

The other 22 rearrangements occurred by unknown mechanisms not involving any repeated sequences, and 11 of these changes resulted in the loss of prophage remnants. Thus, IS elements were the main drivers of large-scale chromosomal rearrangements in these populations. In fact, IS-mediated events accounted for at least half of the rearrangements in the 40,000-generation clones from every population, including all of them in Ara+1 (Fig. S1).

More generally, there were no obvious differences in the distribution of rearrangement types among the twelve populations.

Complex rearrangements. In several cases, we observed genomic rearrangements that appear to have involved multiple successive events. A total of nine inversion events could have generated the complex rearrangements seen in the 40,000-generation clones from populations Ara+1, Ara-3 and Ara-6. A total of three duplications in the clones from populations Ara+2 and Ara+5 were imperfect, with the junctures between the duplicate copies apparently having been deleted following the duplication events (Fig. 1). To evaluate these hypotheses more thoroughly, we analyzed the genome sequences of the corresponding regions in these clones as well as other evolved clones sampled from earlier generations, with a particular focus on the rearrangements observed in population Ara+1.

Optical mapping suggested that the complex rearrangements in population Ara+1 resulted from four successive inversions (Fig. 2). We used PCR to analyze an additional 18

clones from this population including 3 clones sampled at each of 2000, 15,000, 20,000, 30,000, and 35,000 generations, 1 clone sampled at 25,000 generations, and 2 clones (in addition to the one used in the original analysis) sampled at 40,000 generations (Table 1, Table S2). We PCR-amplified each locus affected by each inversion using primer pairs designed for the two borders of the inverted region, both before and after each inversion (Fig. 2, Table S3). We then scored the presence or absence of each inversion in the ancestor and evolved clones based on the presence or absence of the expected PCR products (Fig. 2, Table 1, Table S3). We also sequenced the PCR products to confirm the expected rearrangement boundaries, and these data supported the scenario of four successive inversions, which we will call Inversions 1 through 4 in order of their appearance. Inversion 1 was present in 1 of 3 clones from 2000 generations, in 2 of 3 clones from 15,000 generations, and in all sampled clones from later generations. This inversion was previously reported in a study of changes in the distribution of IS elements in some LTEE populations (33). Inversion 2 extends for ~600 kbp and overlaps ~500 kbp of Inversion 1. Inversion 2 was seen in all of the clones sampled at generations 20,000, 25,000, and 30,000, and in one of the 3 clones from generation 40,000.

All three clones at 35,000 generations and the other two clones at 40,000 generations carried Inversion 1 together with a second rearrangement, likely also an inversion, different from Inversion 2. Thus, PCR assays using the primer pair +1Inv2RF and +1Inv2RR detected the right border of Inversion 2, but no product was obtained using the primer pair +1Inv2LF and +1Inv2LR designed to detect the left border of Inversion 2. None of the evolved clones that we tested had the first three inversions without having all four, with one clone each from generations 30,000 and 40,000 carrying all four inversions. Inversions 3 and 4 have sizes of ~470 and ~260 kbp, respectively. These data together support the scenario in Fig. 2 that outlines the likely chronology of the inversions in population Ara+1. In a similar vein, the optical maps of the clones sampled at 40,000 generations from populations Ara-3 and Ara-6

imply two and three successive inversions, respectively (Fig. S2).

Optical maps suggested three imperfect duplications in the 40,000-generation samples, one in the clone from population Ara+2 and two in the clone from Ara+5 (Fig. 1), with deletions of the conjoined regions between the duplicated copies. The sequence of that region in the Ara+2 clone showed the absence of the junction sequence between the two copies and the presence instead of an IS1 element (Table S1), suggesting the scenario depicted in Fig. 3. Two copies of IS1 likely inserted, in the same orientation, at the end and start of the first and second tandem copies of the duplication, respectively. A subsequent recombination event between these IS1 elements resulted in the deletion of the intervening region, generating the junction seen with the imperfect duplication (Fig. 3, Table S1). The two imperfect duplications in population Ara+5 seem to have occurred by the same mechanism, with deletions of the duplication junctions again being associated with a new IS1 element (Table S1).

Effects of rearrangements on genome size and structure. We analyzed both the optical maps and the genome sequences to estimate the genome size of each evolved clone sampled at 40,000 generations. Genome size was reduced in 10 of the 12 clones by amounts ranging from 0.9% to 3.5% of the ancestral genome size (Fig. 1). However, the clones from populations Ara+4 and Ara+5 showed slight increases in genome size of 0.8% and 0.3%, respectively, that resulted from duplications, including one encompassing ~4% of the genome (~180 kbp) in Ara+4. The overall tendency toward reduced genome size reflected the fact that large deletions were much more common than large duplications.

Two types of structural constraints have been hypothesized to influence genome structure. One hypothesizes a requirement for symmetry between the origin and terminus of replication in a circular chromosome (9,24), and the other is based on the organization of the chromosome into distinct macrodomains (25). Imbalances of less than ~10% in the lengths of the two replichores have been reported to have little or no effect on E. coli growth (25). By contrast, inversions that disrupt the replication terminus macrodomain such that the replication forks meet far from the ancestral replichore junction have negative effects on growth, as do inversions between the replication origin and right macrodomains. The inversion events that we identified in the LTEE populations affect the symmetry of the evolved genomes to various extents (Fig. 4A). Five populations (Ara+3, Ara+4, Ara+5, Ara+6, and Ara-6) show little change; four populations (Ara-2, Ara-3, Ara-4, and Ara-5)

show moderate changes ranging from ~3 to ~5 min of the leading replication branch; and three populations (Ara+1, Ara+2, and Ara-1) show larger changes of ~8 to ~10 min. In Ara+2, one inversion affected both the replication terminus and left macrodomains, while another affected the replication origin and left macrodomains (Fig. 4B). Inversions in Ara-1

and Ara-5 also affected the replication terminus and left macrodomains, while inversions in Ara-2 and Ara-4 affected the replication origin and the adjacent non-structured domains. The four successive inversions in Ara+1 had the most dramatic effects on macrodomain organization, spanning the right replication terminus and left macrodomains. All of the LTEE populations became much more fit than their common ancestor based on competition assays [START_REF] Wiser | Long-term dynamics of adaptation in asexual populations[END_REF], and thus none of these inversions had any highly deleterious effects; however, we do not know whether the inversions were beneficial mutations or, alternatively, were selectively neutral or even weakly deleterious mutations that hitchhiked with other beneficial mutations.

Parallel rearrangements across populations. The optical maps revealed a high level of parallel evolution -that is, similar large-scale rearrangements -across the twelve populations (Fig. 1). We define parallel rearrangements as those chromosomal regions that were affected by the same type of rearrangement event (i.e., deletion, inversion, or duplication) in at least two populations. Based on this criterion, nine distinct chromosomal regions were repeatedly affected by deletions (numbered D1 to D9 from left to right in Fig. 1), three by inversions (called Intervals I1 to I3 in Fig. 1), and one by duplications (numbered D1 in Fig. 1).

Chromosomal region D8 was deleted in all twelve populations (Fig. 1, Tables S1, Table S4), causing the loss of part or all of the rbs operon, which encodes proteins required for growth on ribose. These deletions have been described previously and shown to contribute a small but consistent fitness benefit in the glucose-limited environment of the LTEE (34). Region D4, which encompasses the DNA between the manB and cpsG genes, was deleted in the clones from ten populations (Fig. 1, Table S1, Table S4). In seven of them (Ara+3, Ara+4, Ara-1, Ara-2, Ara-3, Ara-4 and Ara-5), the deletions occurred by recombination directly between these two genes, which share 96% nucleotide identity, resulting in the loss of all 21 intervening genes. In the three other populations (Ara+1, Ara+2, and Ara-6), the deletions occurred by recombination of IS1 elements located between manB and cpsG. In Ara+1 and Ara-6, the deletions involved the ancestral IS1 element present at genome position 2,034,326 and new IS1 copies that had inserted between manB and cpsG, leading to the loss of 18 and 12 genes, respectively; in Ara+2, the deletion occurred between two new copies of IS1 and caused 14 genes to be lost (Table S1, Table S4). Overall, a common set of 12 genes associated with O antigen biosynthesis was lost in all 10 populations affected by these deletions, and a set of six additional genes associated with colanic acid biosynthesis was eliminated in 8 of these populations.

Four of the other repeatedly deleted chromosomal regions D1, D3, D5, and D7 contained prophage remnants (Fig. 1, Table S1, Table S4). The DLP12-like (region D1) and prophage 2 (region D5) loci were each lost in 10 populations, the Qin-like locus (region D3)

was deleted in 6 populations, and the CP-44-like locus (region D7) was lost in 3 populations.

Eleven populations lost either two or three of these prophage regions. Most of these deleted genes have unknown functions. In population Ara-4, the deletion including the DLP12-like prophage was larger and overlapped a deletion found in Ara-2 (region D2 in Fig. 1, Table S4). Region D2 is also affected in population Ara+1.

Region D2 contains several genes that encode proteins involved with the production and regulation of enterobactin, an iron-scavenging siderophore; it was deleted in populations Ara+1, Ara-2, and Ara-4. Region D6, deleted in populations Ara+1, Ara+2, and Ara-6, spans 17 genes including 10 having unknown functions; the other 7 genes are annotated as phage proteins, which suggests that region D6 also corresponds to a phage remnant, although it has not been annotated as such. Region D9 was deleted in populations Ara-3 and Ara-6, resulting in the loss of the hsdSM genes that encode a type 1 restriction-modification complex.

For inversions, we view as parallel changes the chromosomal intervals containing genes that were inverted in at least two populations, regardless of the overall length of the inversions. Three intervals fulfilled this rule (Fig. 1, Table S1, Table S4): I1 in populations Ara+1, Ara+2, Ara-1, and Ara-5; I2 in Ara+2 and Ara-3; and I3 in Ara+2, Ara-2, Ara-4, and Ara-6. Within the I1 interval, a sub-region denoted as 1.1 was affected by smaller inversions in populations Ara+3, Ara-2, Ara-3, and Ara-4; this sub-region was thus inverted in eight of the twelve populations. Sub-region 1.1 contains 148 genes, and this number increases to 1366 over the entire I1 interval. The I2 and I3 intervals contain 345 and 670 genes, respectively. The inversions spanning I1 were mediated by IS1 elements in Ara+2 and Ara-1 and by IS150 elements in Ara+1 and Ara-5, while all of the inversions spanning subregion 1.1 were mediated by IS3 elements. The inversions spanning I2 were mediated by IS150 elements in Ara-3 and by recombination between rRNA-encoding operons in Ara+2.

All of the inversions spanning I3 were mediated by recombination between rRNA-encoding operons.

A single chromosomal region, D1, underwent parallel duplications in three populations, Ara+2, Ara+4, and Ara+5 (Fig. 1, Table S1, Table S4). These three duplications ranged in size from ~11 to ~60 kbp, but they share ~11 kbp and 14 genes (Table S1, Table S4). The rpoS gene that encodes an alternative sigma factor is present in all three duplications, as are pcm and surE, both essential for survival in stationary phase, and the cysDNC operon involved in sulfur metabolism.

Temporal dynamics of rearrangements in population Ara-1. We analyzed the optical maps and genome sequences of eight clones sampled from population Ara-1 at 2000, 5000, 10,000, 15,000, 20,000, 30,000, 40,000, and 50,000 generations. We detected 6 deletion events ranging from ~7 to ~23 kbp in size (Table 2), three of which went to fixation including one that arose before 2000 generations and two that occurred between generations 65 5000 and 10,000. The ~1.5 Mbp inversion that we detected in the 40,000-generation clone (Fig. 1, Table S1) occurred between 5000 and 10,000 generations and was then fixed in the population. Two translocation events were present in the 5000-generation clone but were not seen in any of the later samples. Of the five rearrangements detected at 50,000 generations, four were already present at 10,000 generations (Table 2). Assuming a uniform rate of 5/50,000, one is unlikely to observe 4 or more mutations by generation 10,000 (one-tailed

Poisson test, P = 0.019), suggesting heterogeneity in the evolution of these large-scale rearrangements over time. This heterogeneity might reflect a change in the underlying mutational processes that generate these rearrangements. Alternatively, the rate of fitness improvement and the corresponding rate at which beneficial mutations went to fixation were much higher early in the experiment than later on [START_REF] Wiser | Long-term dynamics of adaptation in asexual populations[END_REF][START_REF] Barrick | Genome evolution and adaptation in a long-term experiment with Escherichia coli[END_REF], and this difference may explain the greater number of rearrangements fixed in the early generations.

Discussion

We combined optical mapping and genome sequencing to identify chromosomal rearrangements that occurred in each of twelve populations over the course of 40,000 generations of experimental evolution. We detected a total of 110 rearrangements, of which 75% were deletions, 17% were inversions, and 8% were duplications; the resolution of the optical mapping did not allow the identification of new IS insertion events. Some of the complex rearrangements were shown to involve a succession of events including multiple inversions as well as duplications followed by deletions overlapping the junction of the two copies. Most rearrangements (~70%) occurred by recombination between IS elements, and many chromosomal regions were repeatedly affected by similar rearrangements in two or more populations. In most populations, the overall chromosomal organization was maintained without a large imbalance of the symmetry between the origin and terminus of replication or any major disruption of the chromosomal macrodomains. However, three populations evolved rather substantial asymmetry between the replication origin and terminus. The dynamics of the rearrangements over time were examined in one population, and they showed that most of the rearrangements were substituted in the early generations of the experiment, when the rate of fitness increase was fastest, suggesting that these rearrangements contributed to the genetic adaptation of these populations.

The twelve experimental populations have been evolving in and adapting to the same environment for tens of thousands of generations. Therefore, one might expect them to lose unused functions and evolve smaller genomes, as observed for bacteria adapting to stable environments, such as endosymbionts adapting to their hosts [START_REF] Ochman | Genes lost and genes found: evolution of bacterial pathogenesis and symbiosis[END_REF][START_REF] Stinear | Reductive evolution and niche adaptation inferred from the genome of Mycobacterium ulcerans, the causative agent of Buruli ulcer[END_REF]. As predicted, deletions were indeed the predominant rearrangements detected in the LTEE. We also observed a high level of parallelism, with nine chromosomal regions deleted in at least two populations (Fig. 1, Table S4). These parallel deletions removed genes from the rbs operon, genes involved in O antigen and colanic acid biosynthesis, and prophage-related genes. In a previous study (34), we demonstrated that rbs deletions occurred at a very high frequency owing to the presence of an IS150 element adjacent to the operon and that they conferred a small but significant fitness increase. More generally, the deleted genes have functions that are not used under the conditions prevailing during the LTEE. These deletions might have conferred higher fitness by eliminating unnecessary and costly gene expression [START_REF] Dekel | Optimality and evolutionary tuning of the expression level of a protein[END_REF], or they might have been effectively neutral if the affected genes were already not expressed. The involvement of IS elements in producing many of these deletions may reflect increased local mutagenesis caused by homologous recombination between two identical elements, similar to the process demonstrated for the rbs operon (34). Deletions have also been reported in evolution experiments with other bacteria and environments (27,38,[START_REF] Nilsson | Bacterial genome size reduction by experimental evolution[END_REF].

In the LTEE, the genome size of the E. coli declined after 40,000 generations in 10 of the 12 populations, with the reductions ranging from 0.9% to 3.5% relative to the ancestor; two populations showed slight increases in genome size of 0.3% and 0.8%. Over all 12 populations, we recorded 70 reductive events with sizes of 1 kbp or larger, with an average of ~17 kbp and a median of ~11 kbp. These values are far below the reductions inferred for pathogenic bacteria including Mycobacterium leprae, Yersinia pestis, and Mycoplasma ulcerans [START_REF] Parkhill | Comparative analysis of the genome sequences of Bordetella pertussis, Bordetella parapertussis and Bordetella bronchiseptica[END_REF]; of course, the time periods over which these pathogens have evolved their reduced genomes are much longer. On a time scale more commensurate with the LTEE, a study of P. aeruginosa adapting to the lungs of human patients with cystic fibrosis found that up to 8% of the ancestral genome was lost over the course of 35 years (38); in that study, the average and median deletion sizes were 44.5 kbp and 26.6 kbp, respectively, for 27 deletion events with sizes of at least 1 kbp. Most deletions in the P. aeruginosa study occurred through illegitimate and homologous recombination events, but IS elements were not involved. By contrast with that dramatic reduction, an analysis of 11 natural isolates of E. coli O157:H7 found only very limited genome reductions of up to 3.7 kbp, or ~0.1%, and slight increases in chromosome size were detected in several of those isolates (39). Note, however, that these estimates exclude the effect of new insertions of IS elements, which are not detected by optical mapping.

Three evolutionary factors have often been suggested as drivers of reduced genomes in pathogenic and endosymbiotic bacteria: severe population bottlenecks, the absence of horizontal gene transfer, and the elimination of selection for various functions owing to the availability of nutrients and other services provided by the host (5,[START_REF] Ochman | Genes lost and genes found: evolution of bacterial pathogenesis and symbiosis[END_REF][START_REF] Stinear | Reductive evolution and niche adaptation inferred from the genome of Mycobacterium ulcerans, the causative agent of Buruli ulcer[END_REF]. Bottlenecks are not severe in the LTEE, with more than 10 6 cells transferred each day to fresh medium. There is no horizontal transfer in the LTEE, as plasmids and functional phage are absent and E. coli

does not undergo natural transformation. The nutritional environment of the LTEE consists of a minimal medium with glucose and ammonium providing carbon and nitrogen, respectively.

Owing to this simple environment, certain functions cannot be lost including, for example, the production of amino-acids. However, some functions are dispensable including those involved with using alternative resources (e.g., the loss of the ability to grow on ribose) and those necessary to thrive in natural environments (e.g., loss of genes involved with O antigen and colanic acid biosynthesis). Thus, the simple flask environment -like a host organismprovides environmental constancy and protection that allow certain functions to be discarded.

In doing so, the cells may save energy, thereby providing a competitive advantage; even without that benefit, any unused functions will tend to decay or be deleted by on-going mutations (20,[START_REF] Cooper | The population genetics of ecological specialization in evolving Escherichia coli populations[END_REF][START_REF] Leiby | Metabolic erosion primarily through mutation accumulation, and not tradeoffs, drives limited evolution of substrate specificity in Escherichia coli[END_REF]. Another factor that contributed to genome reductions in the LTEE is homologous recombination, especially that mediated by IS elements. The majority of rearrangements detected in this study involved IS elements, and these elements often flank non-core genes that were acquired by horizontal gene transfer in the distant past, i.e., prior to E. coli B being brought into the laboratory [START_REF] Studier | Understanding the differences between genome sequences of Escherichia coli B strains REL606 and BL21(DE3) and comparison of the E. coli B and K-12 genomes[END_REF]. These horizontally acquired genes would thus be both dispensable and prone to deletion.

Besides deletions, we detected two other types of large-scale rearrangements, namely duplications and inversions. As for deletions, some of these other rearrangements affected the same chromosomal regions in multiple populations, with that parallel evolution providing indirect evidence that these mutations were beneficial in the context of the LTEE (34,[START_REF] Barrick | Genome evolution and adaptation in a long-term experiment with Escherichia coli[END_REF][START_REF] Crozat | Long-term experimental evolution in Escherichia coli. XII. DNA topology as a key target of selection[END_REF][START_REF] Woods | Tests of parallel molecular evolution in a long-term experiment with Escherichia coli[END_REF]. Duplications were rare, however, with only nine such events detected among the 12 populations after 40,000 generations. The paucity of duplications compared to deletions probably reflects the intrinsic instability of duplications, which readily collapse back to a single copy when cells are propagated under conditions that do not favor having multiple copies of the relevant genes (28). The nine duplications we detected range in size from ~3 to ~180 kbp. Owing to the number and diversity of the genes found in the duplicated regions, it is difficult to know whether and how the duplications affected the fitness of the bacteria.

However, one region was duplicated in three populations, and it spans an ~11-kbp region containing 14 genes including rpoS, which encodes the alternative sigma factor involved in the transition into stationary phase [START_REF] Hengge-Aronis | Survival of hunger and stress: the role of rpoS in early stationary phase gene regulation in E. coli[END_REF]. Previous work has shown that the LTEE populations underwent changes in the regulatory networks involved in the transitions between exponential and stationary phases, which they experience on a daily basis [START_REF] Philippe | Evolution of global regulatory networks during a long-term experiment with Escherichia coli[END_REF][START_REF] Hindré | New insights into bacterial adaptation through in vivo and in silico experimental evolution[END_REF]. The parallel duplications may thus affect the expression of rpoS and thereby confer a competitive advantage during these transitions.

Inversions were the second most common type of large-scale rearrangement seen in the LTEE populations. We detected a total of 19 inversions, of which seven affected more than a quarter of the chromosome, and multiple successive inversions occurred in several populations. Three chromosomal regions were inverted in multiple populations, whereas only one region (spanning genome positions 4,453,625 to 146,102) was not affected by any of the inversions. Owing to the large number of genes in these inversions, it is difficult to predict their effects, if any, on the fitness or other phenotypes of the evolved cells. Chromosomal inversions have been found in natural isolates of many bacteria, including E. coli (39),

Staphylococcus aureus (16,[START_REF] Shukla | Optical mapping reveals a large genetic inversion between two methicillin-resistant Staphylococcus aureus strains[END_REF], Enterococcus faecium [START_REF] Lam | Comparative analysis of the first complete Enterococcus faecium genome[END_REF], Francisella tularensis (13), and Bacillus anthracis [START_REF] Okinaka | An attenuated strain of Bacillus anthracis (CDC 684) has a large chromosomal inversion and altered growth kinetics[END_REF]. Some of these inversions have been related to phenotypic changes, including colony morphology (16) and virulence [START_REF] Okinaka | An attenuated strain of Bacillus anthracis (CDC 684) has a large chromosomal inversion and altered growth kinetics[END_REF], but in most cases their effects are unknown. Experiments have shown that some inversions adversely affect cell growth because they substantially reduce the symmetry between the origin and terminus of replication (24), or because they disrupt the overall organization of the chromosome into macrodomains including especially the structure of the terminus (25). The inversions and other rearrangements that we observed in the evolved genomes of the LTEE had variable effects on the symmetry between the origin and terminus of replication: five populations showed almost no change in symmetry, four had increased asymmetry leading to imbalances of the replication arms of a few percent, and three evolved imbalances of ~8% to ~10% (following the numerical scheme for calculating imbalance used in ref. 25). None of the clones we studied, however, had imbalances as great as the 15% imbalances previously shown to impair cell growth (25). In any case, the variations in chromosomal organization produced by the rearrangements we detected are clearly well tolerated under the conditions of the LTEE. The strong conservation of gene order between E. coli and Salmonella, which began to diverge from one another many tens of millions of years ago, has led to the suggestion that selection places important constraints on gene order (29). In that respect, the extent of inversions observed in the LTEE over two decades is surprising. One possible explanation for this discrepancy is that horizontal gene transfer-in particular, its importance for adaptation to changing environments-generates the constraint. In the LTEE, there is no gene transfer and the experimental environment does not change, relieving the constraint and thus allowing gene order to vary more freely.

An alternative hypothesis is that these inversions may confer higher fitness, perhaps by changing the distribution of genes on the leading and lagging strands. During replication, the DNA and RNA polymerase complexes move along the same DNA molecule, and their physical interactions depend on gene orientation [START_REF] Rocha | Order and disorder in bacterial genomes[END_REF]. Collisions between DNA polymerase and RNA polymerase transcribing genes from the lagging strand occur with a higher probability, thereby causing the replication machinery to stall and potentially also generating truncated transcripts. By contrast, when genes are located on and transcribed from the leading strand, such collisions merely slow down the replication complex and the transcript is released after completion. Thus, essential (as well as highly expressed) genes tend to be located on the leading strand [START_REF] Rocha | Essentiality, not expressiveness, drives gene strand bias in Bacteria[END_REF]. The LTEE ancestral strain has 170 genes that are defined as essential and conserved in 80% of the sequenced bacterial genomes [START_REF] Gerdes | Experimental determination and system level analysis of essential genes in Escherichia coli MG1655[END_REF], and 74% of them are indeed on the leading strand. Using the optical mapping data, we can determine whether the proportion of these genes located on the leading strand changed as a result of inversions in the 40,000-generation clones. Many inversions included either the origin or terminus of replication, and hence they did not affect whether genes were on the leading or lagging strand. However, inversions in three populations (Ara+1, Ara-3, and Ara-6) affected a total of 33 genes, with 27 moving from the leading to the lagging strand and only 6 moving from the lagging to the leading strand. Therefore, these results do not support the hypothesis that the inversions improved fitness by reducing collisions between the DNA and RNA polymerase complexes. However, this negative result may reflect the minimal medium used in the LTEE environment, which limits growth rates and might reduce the importance of these collisions. Also, we cannot exclude the possibility that particular genes might have been subject to this effect, as some essential genes did move from the lagging to the leading strand.

About 70% of the large-scale rearrangements we detected in the evolved clones occurred by homologous recombination between IS elements, and that proportion does not include new insertions of IS elements (because optical mapping cannot resolve such events).

These elements have previously been shown to contribute to evolution in the LTEE in three ways. First, IS elements have generated some beneficial mutations including the deletions of the rbs operon (34). Second, some new IS insertions occurred in genes that were mutated in many or all of the LTEE populations (33,[START_REF] Woods | Tests of parallel molecular evolution in a long-term experiment with Escherichia coli[END_REF], and that genetic parallelism strongly suggests that these insertions were also beneficial. Third, population Ara+1 has undergone a striking increase in IS150 copy number [START_REF] Papadopoulos | Genomic evolution during a 10,000-generation experiment with bacteria[END_REF], including some insertions that can be inferred to be beneficial based on the previous criterion. Here, we have further shown that IS elements, by providing a substrate for homologous recombination, have played the major role in largescale rearrangements that have restructured the genomes during this long-term experiment. IS elements have also been shown to contribute to genomic plasticity in other studies where they were investigated, including both evolution experiments in the laboratory (36,37,[START_REF] Zinser | Bacterial evolution through the selective loss of beneficial genes. Trade-offs in expression involving two loci[END_REF] and analyses of natural isolates (13,[START_REF] Tanaka | An insertion sequence-dependent plasmid rearrangement in Aeromonas salmonicida causes the loss of the type three secretion system[END_REF][START_REF] Mijnendonckx | Insertion sequence elements in Cupriavidus metallidurans CH34: distribution and role in adaptation[END_REF].

In summary, we used optical mapping to find large-scale chromosomal rearrangements that occurred during a long-term evolution experiment with E. coli. The many rearrangements thus discovered had substantial effects on the size and structure of the chromosome, demonstrating the impressive plasticity of bacterial genomes. Several lines of evidence, including parallel changes observed in independently evolving populations, indicate that at least some of the rearrangements conferred higher fitness in the experimental environment. IS elements mediated most of the large-scale rearrangements by providing a substrate for recombination. While new sequencing technologies make it increasingly easy to find point mutations and other small changes in the genomes of experimentally evolving populations, our results demonstrate the value of also analyzing large-scale chromosomal rearrangements in these studies.

Materials and Methods

Bacterial strains. All strains came from the E. coli long-term evolution experiment [START_REF] Wiser | Long-term dynamics of adaptation in asexual populations[END_REF][START_REF] Lenski | Long-term experimental evolution in Escherichia coli. I. Adaptation and divergence during 2,000 generations[END_REF].

Twelve populations, named Ara+1 to Ara+6 and Ara-1 to Ara-6, were founded from the same ancestral strains, REL606 and REL607 (a spontaneous Ara + mutant of REL606). The populations have been propagated by daily transfers in Davis minimal medium containing 25 µg/ml glucose (DM25) as a limiting carbon source [START_REF] Lenski | Long-term experimental evolution in Escherichia coli. I. Adaptation and divergence during 2,000 generations[END_REF]. Samples from each population have been taken at 500-generation intervals and stored at -80°C. For optical mapping, we used one clone isolated from each population at 40,000 generations, as well as one clone from population Ara-1 at each of 2000, 5000, 10,000, 15,000, 20,000, and 50,000 generations.

Additional clones were sampled at several time points from Ara+1 to investigate specific rearrangements. All strains used in this study are listed in Table S2.

Optical Mapping. The optical mapping procedure was performed by OpGen (Gaithersburg, Maryland), as described elsewhere [START_REF] Shukla | Optical mapping reveals a large genetic inversion between two methicillin-resistant Staphylococcus aureus strains[END_REF]. Clones were revived from stocks kept at -80°C in 15% glycerol by overnight growth in LB medium. Genomic DNA was extracted using the OpGen Sample Preparation Kit (OpGen, Inc., MD) and Agencourt Genfind v2 Kit (Beckman Coulter, FL). Single DNA molecules were captured on an Argus ® surface within a MapCard, digested with the NcoI restriction enzyme, and stained with JOJO-1 on the Argus MapCard Processor. They were analyzed by automated fluorescent microscopy using the Argus Optical Mapper. This software records the size and order of restriction fragments for each DNA molecule. Collections of single-molecule restriction maps for each genome were assembled according to overlapping fragment patterns to produce a whole-genome optical map assembly. The consensus optical map assemblies for each evolved clone were then compared to the predicted restriction map of the ancestral strain's genome [START_REF] Jeong | Genome sequences of Escherichia coli B strains REL606 and BL21 (DE3)[END_REF] to identify large-scale rearrangements using the MapSolver software. Rearrangements smaller than ~5 kbp are too small to resolve. Thus, IS insertion events were not detected in this study.

Characterization of rearrangement borders. The precise locations of the rearrangement borders were identified (Table S1) by analyzing the genome sequences of the evolved clones. The genomes of population Ara-1 clones from generations 2000 to 40,000

were previously sequenced [START_REF] Barrick | Genome evolution and adaptation in a long-term experiment with Escherichia coli[END_REF][START_REF] Wielgoss | Mutation rate dynamics in a bacterial population reflect tension between adaptation and genetic load[END_REF], as were those of the 40,000-generation clones from populations Ara-3, Ara-5, Ara-6, Ara+1, Ara+2, Ara+4 and Ara+5 [START_REF] Wielgoss | Mutation rate inferred from synonymous substitutions in a long-term evolution experiment with Escherichia coli[END_REF]. The additional genomes analyzed in this study were the 40,000-generation clones from populations Ara-2, Ara-4, Ara+3, and Ara+6, and they were sequenced on the Illumina Genome Analyzer platform at the Centre National de Séquençage, Genoscope, with one lane of single-end 36bp reads per genome. Sequence reads were compared to the genome of the ancestral strain REL606 (67), using both breseq, a computational pipeline for analyzing resequenced bacterial genomes (35,[START_REF] Barrick | Genome evolution and adaptation in a long-term experiment with Escherichia coli[END_REF][START_REF] Wielgoss | Mutation rate dynamics in a bacterial population reflect tension between adaptation and genetic load[END_REF], and a customized pipeline [START_REF] Vallenet | MicroScope--an integrated microbial resource for the curation and comparative analysis of genomic and metabolic data[END_REF]. The four new genome sequences have been deposited in the European Nucleotide Archive Sequence Read Archive (accession nos. XXXX). The borders of the rearrangements detected by optical mapping were further checked by PCR experiments for two populations, Ara+1 and Ara+2 (Table S1). PCR was performed using 1x reaction buffer, 3 mM MgCl 2 , 0.2 mM dNTP, 0.2 mM of each primer, 50 ng of genomic DNA, and 1.25 unit of Taq DNA polymerase (Invitrogen, Carlsbad, CA) in a 25-µl reaction volume. Reaction mixtures were heated at 95°C for 2 min, then subjected to 32 cycles of 30 s at 94°C, 90 s at 55°C, and 3 min at 72°C, before a final step of 10 min at 72°C. Table S3 lists the primers used to amplify the borders of specific rearrangements. The PCR products were separated by agarose (0.8%) gel electrophoresis in 1x TAE buffer, purified using the Qiagen Gel purification kit, and sequenced (GATC-Biotech, Germany) with the same primers used for PCR assays.

Sequence analysis. Sequences of PCR products containing the borders of rearrangements were analyzed with BioEdit (version 7.0.9.0), and the resulting FASTA files were analyzed using the CLC Sequence Viewer software (v7.0.2, CLC Bio). All sequences were compared to the ancestral genome sequence [START_REF] Jeong | Genome sequences of Escherichia coli B strains REL606 and BL21 (DE3)[END_REF] and checked to confirm the rearrangements deduced from the optical maps. A Python script was written to construct FASTA files containing the reconstructed genome sequences of the evolved clones; we have deposited the script at the Dryad Digital Repository (doi:XXXX). Point mutations and deletions detected in the evolved clones were automatically inserted; other rearrangements were added by hand in CLC Sequence Viewer. The leading strand branch length was calculated from the origin of replication oriC [START_REF] Kaguni | Replication initiation at the Escherichia coli chromosomal origin[END_REF] to the middle of the terminus region defined by the dif locus [START_REF] Ip | Decatenation of DNA circles by FtsKdependent Xer site-specific recombination[END_REF]. For the evolved clones, the branch-length measurements were based on the rearranged genomes using the new locations of these two loci. in Ara-3) have sizes larger than one half of the chromosome (Table S1). These inversions could have been described alternatively as inversions of the other part of the chromosome, with their sizes then being smaller than one half of the chromosome. For example, we describe inversion 1 as being ~2.8 Mbp, whereas according to the alternative its size would be ~1.8 Mbp. We use the coordinates according to the ancestral map for internal consistency, and this choice does not affect any conclusions. within I1, were affected repeatedly by inversions. We describe the boundaries of all chromosomal rearrangements according to the ancestral map shown here. As a consequence, three inversions (inversion 1 in Ara+1, inversions 1 and 2 in Ara-3) have sizes larger than one half of the chromosome (Table S1). These inversions could have been described

alternatively as inversions of the other part of the chromosome, with their sizes then being smaller than one half of the chromosome. For example, we describe inversion 1 as being ~2.8

Mbp, whereas according to the alternative its size would be ~1.8 Mbp. We use the coordinates according to the ancestral map for internal consistency, and this choice does not affect any conclusions. wbbD, wbbC, wzy, wbbB, wbbA, vioB, vioA, wzx, rmlC, rfbA, rfbD, rfbB, galF, wcaM, wcaL, wcaK, wzxC wbbD, wbbC, wzy, wbbB, wbbA, vioB, vioA, wzx, rmlC, rfbA, rfbD, rfbB, galF, cpsG manB, manC, insB-14, insA-14, wbbD, wbbC, wzy, wbbB, wbbA, vioB, vioA, wzx, rmlC, rfbA, rfbD, rfbB, galF, wcaM, wcaL, wcaK, wzxC cpsG manB, manC, insB-14, insA-14, wbbD, wbbC, wzy, wbbB, wbbA, vioB, vioA, wzx, rmlC, rfbA, rfbD, rfbB, galF, wcaM, wcaL, wcaK, wzxC yjcF, actP, yjcH, acs, nrfA, nrfB, nrfC, nrfD, nrfE, nrfF, nrfG, gltP, yjcO, fdhF, yjcP, yjcQ, yjcR, yjcS, alsK, alsE, alsC, alsA cpsG manB, manC, insB-14, insA-14, wbbD, wbbC, wzy, wbbB, wbbA, vioB, vioA, wzx, rmlC, rfbA, rfbD, rfbB, galF, wcaM, wcaL, wcaK, wzxC hokE, insL-3, entD, fepA, fes, ybdZ, entF, fepE, fepC, fepG, fepD, ybdA, fepB, entC, entE, entB, entA, ybdB, cstA, ybdD, ybdH, ybdL, ybdM, cpsG manB, manC, insB-14, insA-14, wbbD, wbbC, wzy, wbbB, wbbA, vioB, vioA, wzx, rmlC, rfbA, rfbD, rfbB, galF, wcaM, wcaL, wcaK, wzxC acpD, hrpA, ydcF, aldA, gapC, insA-12, insB-12, cybB, ydcA, hokB, cpsG manB, manC, insB-14, insA-14, wbbD, wbbC, wzy, wbbB, wbbA, vioB, vioA, wzx, rmlC, rfbA, rfbD, rfbB, galF, wcaM, wcaL, wcaK, wzxC cpsG manB, manC, insB-14, insA-14, wbbD, wbbC, wzy, wbbB, wbbA, vioB, vioA, wzx, rmlC, rfbA, rfbD, rfbB, galF, wcaM, wcaL, wcaK, wzxC cpsG manB, manC, insB-14, insA-14, wbbD, wbbC, wzy, wbbB, wbbA, vioB, vioA, wzx, rmlC, rfbA, rfbD, rfbB, galF, wcaM, wcaL, wcaK, wzxC yjiV, mcrC, mcrB, yjiW, hsdS, hsdM, hsdR, mrr, yjiA, yjiX, yjiY, hpaC, hpaB, hpaA, hpaX a All positions are given according to the genomic coordinates of the ancestral strain [START_REF] Jeong | Genome sequences of Escherichia coli B strains REL606 and BL21 (DE3)[END_REF]. IS insertion events are not reported because optical mapping cannot resolve them.

b RRE, recombination between repeated elements, with the identity of the repeated element indicated after the hyphen.

c For rearrangements involving more than 25 genes, only the first and last genes altered by the rearrangement are shown, along with the total number of genes affected. Prophage regions are also indicated.

d Rearrangements involving chromosomal regions that evolved in parallel (at least two populations) are indicated based on the designations shown in Fig. 1.

e These rearrangements involved multiple successive events. The first event in a series gives the location of the altered region relative to the ancestral genome. For the later events in the series, the indicated coordinates reflect the altered region compared to the preceding state in that series.

f Specific type of successive events, in which a duplication was followed by a deletion of the junction between the duplicated copies. The indicated size of the duplication is the one that existed before the deletion occurred. The rearrangements are numbered according to their location from left to right in Fig. 1 and, for the four successive inversions in Ara+1, in their chronological order (Fig. 2). a The regions are numbered according to Fig. 1. The range of genomic coordinates for each region are shown in bold font, with positions given according to the genomic coordinates of the ancestral strain (67).

b All positions are given according to the genomic coordinates of the ancestral strain the ancestor genome (67).

c RRE, recombination between repeated elements, with the identity of the repeated element indicated after the hyphen.

d For parallel inversions, we give the name of the first and last genes affected followed by the total number of genes included in each inversion.

For multiple inversions, we use the same rules to describe their coordinates as used in Table S1.

e The sizes of these deletions vary among the populations, and only some of them eliminate the entire operon (34).

Feedback from the Editor of mBio and two anonymous reviewers

Re: mBio01377-14 (Large Chromosomal Rearrangements during a Long-Term Evolution Experiment with Escherichia coli) Dear Dr. Schneider:

We have completed our review of your manuscript, and I am pleased to inform you that, in principle, we expect to accept it for publication in mBio. However, acceptance will not be final until you have addressed the reviewer comments

The editor who handled your manuscript thinks that the modifications required are minor and that the work can be accepted if the reviewer comments are adequately addressed Thank you for the privilege of reviewing your work. Below you will find instructions from the mBio editorial office and comments generated during the review.

Sincerely,

Arturo Casadevall

Editor in Chief, mBio mBioEditorInChief@asmusa.org (Signing for the editors)

Editor comments:

The mBio Editor in charge fully agrees with the decision of the Invited Editor for this manuscript. He would like to comment few points to be considered by the authors to produce the final version:

1. A main message of the manuscript is that a large number of rearrangements occurs along the long-term experimental evolution experiment, influencing the size and structure of the chromosome, "demonstrating the impressive plasticity of bacterial genomes" (line 21).

However, in many microorganisms, bacterial clones isolated along many years in different places, probably resulting from very, very long-term natural evolution processes, show a remarkable chromosomal stability, in spite of exposure to different environments that could eventually select for particular conformations. In the author's classic experiment, the conditions of experimental propagation are fairly constant, and, unexpectedly, heavy plasticity is found. Might be the authors would like to include a comment this paradox, helping the reader to understand the possible generality of their findings. Additionally, such a comment would certainly satisfy Reviewer 1, who is asking for better explanation about the "beneficial effects" of evolved chromosomal changes.

2. Line 197: "Six of the 12 populations had evolved hypermutable phenotypes", however (line 200-202) "there is no suggestion of any difference in the overall rate at which rearrangements accumulated in mutator versus non-mutator populations". To the knowledge of this Editor, a recent paper (Turrientes MC et al. 2013. Normal Mutation Rate Variants Arise in a Mutator (Mut S) Escherichia coli Population. PLoS ONE 8( 9): e72963. doi:10.1371/journal.pone.0072963) found in a clinical E. coli mutator clinical strain submitted to serial passages, a high frequency of transposition events, and different inversionpatterns of chromosomal fragments were detected in evolved variants.

3. Lines 300-303: "Region Δ4, which encompasses the DNA between the manB and cpsG genes", "deletions occurred by recombination directly between these two genes, which share 96% nucleotide identity, resulting in the loss of all intervening genes". In occasions manB is confused with cspG (http://www.uniprot.org/uniprot/P24175), and there is "another" cpsG (cold shock protein G) gene unrelated with manB. Clarify.

4. Line 311. Probably "colanic", not "colonic" acid. and genomic stability could be appropriate -is the "long-term evolution experiment" performed by the authors a stressful experience for E. coli?

Invited Editor comments:

Your manuscript on "Large chromosomal rearrangements..." has now been reviewed by two experts in the field and myself. There is a general agreement that the results presented in your manuscript have general interest and are important as reference for evolutionary experiments, but as commented by reviewer 1 the text could be shortened considerable, which would help focusing on the parts with general information to the readers. Both reviewers point out important things which you should address carefully.

Reviewer #1 (Comments for the Author)

The authors describe the use of whole-genome optical mapping to identify large-scale chromosomal rearrangements in the genomes of E. coli that are part of a long-term evolution experiment.

The experiment is an important one, and these results are likely to be of interest, however the manuscript is very long and dry, with the minutiae of the rearrangements spelt out in great detail. I believe that the overview and general import of the results are likely to be of broad interest, but the fine details are very specific to this set of experimental conditions, and are likely to be of interest only to those who are invested in this particular experiment.

Specific points:

There is a tendency to see, or imply, selection and functional effects underlying each change, or at least to put this forward as the first explanation.

For example: I have some concerns about the discussion of inversions, which starts out with quite strong statements, but is heavily caveated towards the end of the discussion. At line 190, the authors state "In seven cases ... more than a quarter of the chromosome was affected ...". Can you really say that all these genes are "affected" by the inversion in any meaningful way? As the authors themselves admit later on, the vast majority of these genes will have no meaningful change in local or global context (as most are reciprocal around the origin or terminus), and are unlikely to be affected in any way. Therefore the fact that they are affected in multiple strains may be simply chance (especially given the size of the inversions). I think this needs to be re-worded in a more neutral way.

In lines 477-481, the authors describe the apparent lack of constraint on gene order in the LTEE, suggesting that horizontal transfer may generate this constraint in the wild. Another possibility is that selection on gene order is weak, and only effective over very long timescales. A similar example would be selection for codon usage -this is clearly apparent over very long timescales, but there is no evidence it is detectable on the timescale of the LTEE.

Lines 316-317, discussing prophage deletions: "Most of these genes have unknown functions". Given that they are within prophage, it's highly likely that most of these genes have no function at all for the E. coli.

Lines 338. Seeing repeated inversions between rRNA genes is not surprising -these are frequent in nature (certainly in Salmonella), and given the size of the perfect repeats, they may just happen more frequently than other rearrangements, and thus be seen more often.

All these points belie the statement in line 439 (which recurs throughout the manuscript), that events affecting multiple locations provide "indirect evidence that these mutations were beneficial". This may not be true -they may just be very large and neutral, or smaller and simply occur more frequently, and this needs to be discussed more rigorously.

Reviewer #2 (Comments for the Author):

The paper provides a detailed analysis of all large chromosomal rearrangements that have appeared in 12 independently evolving populations of E. coli propagated in shake-flasks for ~40, 000 generations.

The investigations on the 12 populations have already led to numerous papers addressing questions in evolutionary biology, and as such the experiment constitutes an important point of reference within the field of experimental microbial evolution. While genome sequences of clones from eight of the 12 populations have previously been analyzed by Barrick et al. (Nature 461(7268), 2009) and Wielgoss et al. (G3 1(3), 2011), this is the first whole-genome comparison that include clones from all 12 populations. Furthermore, as the authors choose to focus their analysis on large chromosomal rearrangements, they use optical mapping on genomes from all 12 populations to complement the short-read whole-genome sequence data, and in particular the optimal mapping enables the identification of inversions involving long sequence repeats.

All together, this is an immense amount of genomic data, but the authors manage to present it in a clear and efficient manner. Accordingly, the study is successful in giving a general overview of large chromosomal rearrangements that have accumulated in clones from the 12 populations after 40,000 generations of growth. Also, genomic information of longitudinal collected isolates from some of the populations is analyzed to determine the succession and dynamics of rearrangements over time. Finally, the authors identify a high level of parallel evolution across the populations, and this suggests that these rearrangements have been positively selected. Some of the findings have already been reported previously (for example parallel loss of the rbs operon), but the authors are clear and transparent about this.

Overall, the study gives an overview of large chromosomal rearrangements occurring in a long-term evolution experiment, and its findings may become of relevance as more studies employ similar experimental and analytical techniques to explore genetic variation beyond identification of point mutations. However, the study is very descriptive in its nature, and many of the hypotheses are not substantiated by further experiments.

The text reads nicely, and evolutionary theory is well presented.

Additional suggestions and comments:

(1) Line 175: It is not possible to identify IS element insertions using optical mapping.

However, why are the short Illumina reads not used to detect such events? Barrick et al. uses the exact same type of data (36-nt single-end reads) to identify 10 IS element insertions in the Ara-1 population (Nature 461(7268)).

(2) Line 177: The study concerns large deletions and insertions. However, it is not stated what is the lower limit of "large" deletions and insertions? For example, would a 300-bp deletion be reported in this study?

(3) Line 248: There are three cases of imperfect duplications with deletions of the conjoined regions between duplicated copies. This is an interesting observation, could authors speculate about possible selective advantages from such genetic constructions? In line 441 authors discuss the intrinsic instability of duplications -could it be that the imperfect duplications are more stabely maintained in the chromosome? 121 (4) Line 458: It is understandable that it is difficult to predict the fitness of the inversions due to the large number of affected genes. Nonetheless, in some cases it might be relevant to focus on the genes at the flanks of the inversions in cases where the promoters (or other regulatory elements) of genes are replaced. It could be interesting if it could be predicted that some of the inversions changed transcriptional regulation of genes? Please return the manuscript within 30 days; if you cannot complete the modification within this time period, please contact the editorial office at mbio@asmusa.org. If you do not wish to modify the manuscript and prefer to submit it to another journal, please notify the editorial office of your decision immediately so that the manuscript may be withdrawn from consideration by mBio.

Since your manuscript is likely to be accepted, please be sure to upload all of your native files 122 (i.e., not a single PDF) in production-ready formats. You can check your files using the Rapid Inspector tool at http://rapidinspector.cadmus.com/RapidInspector/zmw/index.jsp, and you can direct questions to our digital art specialists at figures@asmusa.org. This will help us avoid processing delays and move quickly to online publication of your important work.

Please contact us if you have any questions.

Maisha Miles

Managing Editor, mBio

Rob Arthur

Assistant Managing Editor, mBio mBio@asmusa.org American Society for Microbiology Insertion Sequence (IS) elements are bacterial mobile genetic elements that generate diversity by either moving within genomes or being substrates for homologous recombination. Their distribution is highly variable in bacterial species and IS-related mutations confer detrimental to beneficial fitness effects. Invasion of genomes by IS elements is driven by evolutionary forces that are mostly unknown, but likely involving a balance between these contrasting effects. We investigated the evolutionary dynamics of IS elements during long-term evolution with Escherichia coli. Genome invasion by the IS150 type of element is driven by its fitnessenhancing effects, followed later during evolution by a reduction of transposition activity, most likely owing to a higher proportion of deleterious mutations when cells are well adapted to their environment.

Microbial organisms evolved several mechanisms, including the ability to produce mutations, the global regulation of gene expression, the activity of mobile genetic elements and genetic exchange, to both promote phenotypic and genetic diversity and adapt to diverse environments. The pace of evolutionary processes involves, both at short and longer time scales, a balance between evolvability and robustness (1-4) that manifests itself in the finetuned dynamics of the molecular pathways underlying these mechanisms. While this balance has been deeply investigated in the case of mutation rates and regulatory networks, it is less well understood for the dynamics of mobile genetic elements. For instance, the forces driving the ability of bacterial Insertion Sequence (IS) elements to colonize genomes over long evolutionary times are still mostly unknown.

Both theory and experiments have shown that mutation rates are highly dynamic. A high proportion of hypermutator strains, owing to defects in DNA repair genes, evolved both in clinical settings and during propagation in the laboratory (5)(6)(7)(8)(9)(10). By producing a higher number of beneficial mutations, hypermutators increase in frequency in populations subject to changing environments (11,12). However, they also increase the genetic load by producing more deleterious mutations. Hence, after a first rise of mutation rates, compensatory processes can occur to decrease mutation rates resulting in higher fitness once populations are adapted (4,13).

Global regulatory and metabolic networks have highly dynamic topologies that allow exquisite and fast adjustment of genome expression after environmental challenges (14). Both microbial evolution experiments and natural isolates revealed that cellular networks are evolvable at a longer time scale, being rewired after the occurrence of mutations that increased organismal fitness (15). Many theoretical and empirical studies highlighted the pleiotropy, epistasis, plasticity and modularity of these networks. In particular, modularity can provide robustness when pleiotropic effects are deleterious (16). Therefore, both mutation rates and cellular networks result in a balance between stability and evolvability.

IS elements are small (~0.7 to ~2.5 kbp) and highly diverse mobile genetic elements, present in almost all bacterial genomes (17). All IS elements encode functions devoted only to their transposition and its regulation, including one to three Open Reading Frames producing the transposase enzyme and in most cases terminal inverted repeats (IRs). Upon insertion, they can generate a direct duplication of a few base pairs of their target site. Both their activity and presence produce major effects on bacterial fitness because they impact on genome structure and expression (18,19). Indeed, IS transposition can inactivate gene expression but also result in gene deregulation owing to the presence at their extremities of outwardly directed promoters or transcriptional regulatory elements. In addition, homologous recombination between IS copies can produce large chromosomal rearrangements (inversions, deletions, duplications). IS elements are characterized by a very high variability in their type and frequency, as well as in the distribution of different IS families and the copy number of individual IS elements, among bacterial genomes (20). They can have two conflicting effects on bacterial fitness, thereby resulting in debates about their evolutionary significance. First, transposition of IS elements can have detrimental effects and are considered as genomic parasites, maintained mainly through high rates of transposition and acquisition by horizontal transfer (21). As a consequence, transposition is highly regulated by both the element itself and host factors (18). Second, IS elements may generate genetic diversity on which natural selection can act. During laboratory evolution experiments, IS elements have been shown to produce fitness-enhancing mutations under diverse environmental conditions (22)(23)(24)(25)(26)(27), and may therefore be under positive selection. Moreover, IS elements have been suggested to be involved in reductive genome evolution (28).

However, it is mostly unknown whether and how the tension between the detrimental and beneficial effects of IS elements can impact on the dynamics of their copy number distribution inside genomes of single species. One reason is that the long-term dynamics of IS copy number during organismal evolution is difficult to analyze.

To address these questions, we used the longest-ongoing evolution experiment in which 12 independent populations have been founded from a common E. coli ancestor and propagated for over 60,000 generations in a defined environment with a limited amount of glucose as a carbon source (29). Two populations were previously analyzed for the IS distribution after 10,000 generations, revealing an unexplained strong increase in copy number of the IS150 element in one of them (30). Here, we investigated the distribution, dynamics, regulation and overall mutagenesis contribution of IS elements in the 12 populations during 40,000 generations. We found a specific and strong increase in IS150 copy number in one population, called Ara+1, in which IS elements accounted for ~50% of all genomic mutations. We investigated three scenarios for this high copy number increase: mutational alteration of IS150 itself, deregulation of transposition, and fitness-related effects.

Both experiments and a simulation model revealed that Darwinian selection accounted for the copy number dynamics, followed by changes in transposition regulation most likely due to a higher number of deleterious events later on when bacterial cells are adapted to their environment.

Results and Discussion

Distribution of IS elements after 40,000 generations of evolution. We previously examined the distribution of the seven types of IS elements present in the ancestral genome (IS1, IS2, IS3, IS4, IS30, IS150 and IS186) in two populations (Ara-1 and Ara+1) after 10,000 generations (30). Here, we compared the IS fingerprints of 28 evolved clones sampled mutator populations, and only Ara+1 caused a distortion toward a higher ratio (Fig. S3).

These data may suggest differential activities of IS elements in Ara+1.

Dynamics of IS-related changes in Ara+1 over time. The distribution of IS-related changes in Ara+1 was almost similar when comparing the genome sequences and IS fingerprints (Fig. 1). We therefore scrutinized the genome sequences of 20 evolved clones sampled over time from population Ara+1 (Table S1) to determine the number of IS-related changes and their contribution relative to the total mutations (Fig. 2). The number of IS-related changes increased almost linearly over time, with most changes attributed to IS150 (Fig. 2A). The proportion of IS-related changes relative to total mutations was very high and dynamic, increasing strongly to over 50% during the first 5000 generations and then stabilizing (Fig. 2B). Again, most of this dynamics involved IS150.

Three non-exclusive hypotheses may explain this strong increase in IS150 copies during evolution: changes in the IS150 sequence may produce an over-active copy; changes in IS150 regulation owing to mutations substituted during Ara+1 evolution may increase its transposition frequency; initial IS150 transposition events may enhance cell fitness resulting in subsequent increased copy number and thereby transposase amount.

Sequence and transposition frequencies of IS150 during evolution of Ara+1. We scrutinized the genome sequences of the 40,000-generation clones from population Ara+1 (Table S1) and found neither point mutations nor indels inside IS150. Moreover, we PCRamplified and sequenced 19 of the 29 new IS150 copies from the evolved clone 11008 using primers complementary to their flanking regions (Table S4). We found no changes compared to the ancestral copy.

We measured transposition frequencies of IS150 by introducing the reporter plasmid pFDX2339 (33) in the ancestor and five evolved clones from population Ara+1 (Table S1):

one at 2000 (1158C), one at 20,000 (9282B), and three at 40,000 generations (11008, 11009, and 11010). This thermosensitive plasmid carries a modified IS150 element and allows to estimate the frequency of both recombination between the plasmid-borne IS150 and any chromosomal copy, which we used as a proxy to account for the increase in IS150 copy number, and IS150 transposition from the plasmid into the chromosome. As expected, recombination frequencies increased over time with IS150 chromosomal copies (Fig. 3A).

Although transposition frequencies increased by ~50-fold up to 20,000 generations, it likely reflected the copy number increase, and therefore the higher transposase amount, since the proportion of transposition events relative to recombination events was stable (Fig. 3B).

Therefore, we detected no measurable changes in the regulation of IS150 transposition during 20,000 generations of evolution in Ara+1 that may explain the strong increase in IS150 copy number. The mutations that were substituted during 20,000 generations did therefore not interfere with IS150 transposition.

In striking contrast, IS150 transposition frequencies relative to recombination decreased by 2 to 10 fold in all three 40,000-generation clones (Fig. 3B). Therefore, after the initial strong increase in IS150 copy number, transposition decreased later on, implying that mutations substituted between 20,000 and 40,000 generations may affect transposition and likely reflecting detrimental effects of new IS150 copies. Hence, after 20,000 generations, the IS150 copy number is very high and cells are adapted to their environment. Any new transposition events may therefore produce deleterious mutations. This transposition dynamics is reminiscent to what was observed for point mutation rates (4,13). S5). One of the two deletions removed rbs genes involved in ribose consumption and was shown to be beneficial in these conditions (25). (A similar situation was found in another 2000-generation clone called 1158A, Table S5). The new insertion IS150::ybeB was previously shown to confer a fitness increase of 1.176 (34, H 0 = 1; t-test, t s = 18.0391; P = 0.0001). The two other IS150 insertions occurred within nadR and hokB. We constructed a deletion of each of these genes within the Ara-ancestral chromosome, and competed each mutant strain against an Ara+ derivative of the ancestor. These competition assays showed that inactivation of nadR conferred a fitness benefit of 1.14 relative to the ancestral allele (H 0 = 1, t-test, t s = 4.9880, P = 0.0076), while inactivation of hokB was neutral (H 0 = 1, t-test, t s = 0.4090, P = 0.6920). The sum of the fitness effects of both nadR and pbpA evolved alleles accounted for the estimated fitness of the clone 1158C (34, 1.243, H 0 = 1; t-test, t s = 15.0853; P < 0.0001). Thus, the initial new IS150 copies conferred very high fitness to the evolved clones, and the initial copy number increase from 5 in the ancestor to 8 after 2000 generations was likely driving the later increase. The initial copy number increase may produce IS150 transposase levels high enough to further increase transposition activity (33).

Simulation of the dynamics of IS elements. We constructed an evolution model that includes both point mutations and IS-related changes. One hundred replicate populations were propagated for 20,000 generations under each of 18 parameter sets: three mutation rates (μ m ) similar to those in the long-term evolution experiment (4), three rates of IS-related changes (μ IS ) for each mutation rate, and impact or not of IS elements on their transposition rates in each case. Without impact of IS elements on transposition, their proportion relative to point mutations after 20,000 generations were as expected from their initial transposition rates (Fig. 4). By including an impact, no significant contribution of IS-related changes relative to point mutations was detected at the lowest μ IS (0.2) whereas a significant difference was observed at higher μ IS (0.3) for one of the three μ m and for all three μ m at the highest transposition rate (0.5), thereby only at high initial μ IS .

We analyzed into deeper details the simulation with μ m = 2 × 10 -5 and μ IS = 0.3 (Fig. S4). Whether IS elements impact or not on μ IS , all 100 replicate populations revealed similar fitness trajectories, mimicking the 12 long-term E. coli populations (29). Three of the 100 replicate populations revealed both a higher number of IS-related changes including a gradual increase of these changes and a higher proportion of IS-related changes relative to total mutations after 20,000 generations that started early during evolution (Fig. S5, P = 0.002), reminiscent to the IS trajectories in population Ara+1 (Fig. 2). We investigated the fitness effect of the five initial mutations, including 1 point mutation and 4 IS-related changes for each of the three populations. Not only were all IS-related changes beneficial, but they also conferred over the three populations 85% of the fitness increase after the first 5 mutations (and 82% after 20,000 generations), for an average of 36% over all replicates as expected for μ IS = 0.3. In 84 other populations, the 5 initial mutations also included IS insertions, but less than four in all cases. One single exception was observed with one replicate population showing 4 IS-related changes among the 5 initial mutations without a later massive IS expansion. However, IS-related changes accounted for 60% of the mutations in this replicate.

In this case, IS-related changes conferred 74% of the fitness increase after the first 5 mutations (and 70% after 20,000 generations).

Although simple, this model suggests that an initial increase of IS elements, selected owing to their positive effect on fitness, can drive a highly dynamic IS trajectory with a strong expansion during later evolution without any changes in either the IS nucleotide sequences or their regulation. It also suggests a threshold level in both the initial copy number increase and fitness effect above which the later expansion can occur.

the initial evolution steps was likely driven by the fitness-enhancing effect of the first IS copies, followed much later by a reduced transposition activity to compensate for the deleterious effects of further transposition events. We hypothesize that this transposition compensation may result from changes in transposition regulation owing to mutations substituted during the evolution of the Ara+1 population. Whether these mutations were selected for their direct effects on transposition or other effects, with transposition reduction being merely an associated trait, is unknown. The population Ara+1, although retaining the low ancestral mutation rate, can be considered as a mutator population owing to the dynamics of IS150.

The long-term evolution experiment provided enough time to observe the dynamics of IS elements, and in particular how they can initially invade bacterial genomes and be domesticated later on.

Materials and Methods

Bacterial strains and culture conditions. Twelve populations, called Ara+1 to Ara+6 and Ara-1 to Ara-6, were founded from a common ancestor of E. coli, REL606 (35), and serially propagated for over 60,000 generations in a minimal medium (DM25) containing 25 µg/mL glucose (36). Samples from each population were collected at 500-generation intervals and stored at -80°C. Individual clones can be isolated directly from these frozen fossil samples after plating on solid medium. Here, we used the ancestor, two evolved clones sampled from each population after 40,000 generations, two additional clones from Ara-2, one additional clone from Ara+1, Ara-1 and Ara-5, and two clones from Ara+1 at each of the time points 500, 1000, 1500, 2000, 5000, 10,000, 15,000, 20,000, and 30,000 generations (Table S1). All strains were grown in LB liquid cultures or onto LB-agar (12 g/L) plates supplemented when relevant with kanamycin (Kan, 30 µg/mL) and/or chloramphenicol (Cam, 40 µg/mL).

Selection for sucrose-resistant clones during allelic exchange experiments was performed by plating cells onto sucrose plates in which 5% sucrose but no NaCl was added to LB-agar. To distinguish Ara-from Ara+ clones, cells were spread onto tetrazolium-arabinose indicator plates (34).

IS fingerprinting. IS fingerprints were obtained for a total of 29 strains: the ancestor REL606 and 28 evolved clones that were sampled from the 12 populations at 40,000 generations (Table S1). Strains were grown in LB medium for 12 h at 37 °C. Their genomic DNA was extracted by standard methods and quantified spectrophotometrically. Genomic DNA (5 µg) was digested by restriction enzymes that do not cut within the DNA fragments used as probes for each IS element: EcoRV for the fingerprints of IS1, IS2, IS3, IS30 and IS186, and HincII and PvuII in the case of IS4 and IS150, respectively. The resulting fragments were separated by agarose gel electrophoresis and transferred onto nylon membrane. Southern blot hybridizations were performed at high stringency (68 °C) using internal fragments of IS elements as probes. Probes were produced by PCR of REL606 genomic DNA using primer pairs specific to each IS element (37), and then cold-labelled using the DIG system (Roche) according to the manufacturer's protocol. IS fingerprints were obtained after immuno-detection using an anti-DIG antibody, washing steps and exposition of the membranes to autoradiography films. Each IS copy appears as a hybridizing-band and the 29 strains were scored for the presence or absence of each copy. We detected a total of 210 unique IS copies yielding therefore a matrix of 29 strains and 210 fragments over the seven IS elements (Table S2).

Phylogenetic analysis.

The phylogeny of all evolved clones was constructed as a Neighbour-Joining tree (38), with the root being the ancestor, and plotted using the DRAWGRAM program (39). Genetic distances were computed as the total number of differences between each evolved clone and their ancestor. Losses and gains of IS copies have equal weights and simultaneous loss and gain of hybridizing bands from single mutational events were not accounted for.

PCR experiments for probe design and sequencing of new IS150 copies. Polymerase chain reaction (PCR) was performed in a 25-µL reaction buffer containing 3mM MgCl 2 , 0.2mM dNTP, 0.2mM of each primer and 1.25 unit of Taq polymerase. The PCR products were separated by agarose gel electrophoresis, purified using the Qiagen Gel purification kit (Qiagen) and sequenced (GATC Biotech) with the same primers. We used primer pairs to construct the probes specific to all IS elements (37) or sequence new copies of IS150 (Table S4). All sequences were compared to the IS150 sequence present in the genome of the ancestor REL606 (35).

Measure of the transposition frequencies of IS150. Cells from the ancestor and five evolved clones from population Ara+1 (1158C, 9282B, 11008, 11009 and 11010, Table S1)

were electro-transformed with the pFDX2339 reporter plasmid (33) using the GenePulser II equipment (BioRad) according to standard procedures. This plasmid carries a thermosensitive replication origin, allowing its maintenance at low but not high temperatures, a gene conferring Kan resistance for selection of plasmid-carrying cells, and a modified IS150 element bearing a gene conferring Cam resistance (33). At high temperature, three scenarios may happen: first, the plasmid and its modified IS150 may be lost; second, the entire plasmid may integrate in the chromosome by recombination between its own IS150 copy and one chromosomal IS150; third, the plasmid-borne IS150 may transpose into the chromosome while the rest of the plasmid is lost. Each alternative can be distinguished by plating cells onto different selective media.

accggtcgattctgataagc-3')/G236

(5'-cagaaagtcgacaggcacccg-3') and G126

(5'agtcgtggcatgaaacgctg-3')/G237 (5'-cgcgcagtcgactagcagattc-3'), respectively, and fuse them to generate the deleted allele. For nadR, we PCR-amplified nadR with its adjacent regions with primer pair G122 (5'-gtctggcaatcctgctggct-3')/G153 (5'-ggtcacccgtgcaggttt-3'),

followed by a NruI/HincII digestion which generates a 522-bp nadR in-frame deletion.

Pairwise competition experiments were performed to estimate the fitness effects of the deleted nadR and hokB alleles. In all cases, the competing strains also differed by the neutral Ara marker, which allowed them to be distinguished by colony color on tetrazoliumarabinose plates. To ascertain the effect of the deleted alleles, the Ara+ variant of the ancestral strain, REL607, was used in competition experiments against each of the constructed strain. The fitness of one competitor relative to the other was calculated as the ratio of their net growth rates during competition (36). We used t-tests to evaluate whether the mean fitness differed from the null hypothetical expectation of 1 for each set of assays. with effects that are governed by random selection (Table 1). Organisms with higher F have a faster doubling time and can invade the population. We included in our model point mutations occurring at a rate μ m and IS insertions occurring at a transposition rate μ IS divided by the number of initial IS elements that was set to 5 which corresponds to the ancestral copy number of IS150. Values of mutation and transposition rates were varied: μ m was set to either 1.5 x 10 -5 , 2 x 10 -5 or 3 x 10 -5 and μ IS to ratios of 0.2, 0.3 and 0.5 for each μ m value, resulting in a total of 9 different parameter sets. In addition, the model allows, or not, IS elements to increase their transposition rate (μ IS / n), where n is the IS initial copy number. One hundred replicate populations were propagated with each parameter set, leading therefore to a total of 1800 populations (100 x 9 parameters x 2 IS insertion effects). Each population was sampled at 100-generation intervals for 20,000 generations and, for both the entire population and the dominant sub-population, we scored the number of mutations, number of new IS copies, mutation order, and F. A B Moreover, these results illustrate the power of using experimental evolution experiments to perform in depth analysis of evolution. Our tool, the Long Term Evolution Experiment (LTEE), allows for an exhaustive approach as we can retrace almost every step undertaken by evolution due to easy access to a revivable fossil record. Classical studies of evolution are based on the observation of clones that have diverged for a long evolutionary time, focusing only on the surviving and then successful mutants and using a highly incomplete and imperfect fossil record.

Model of evolution

"I endeavoured, also, to show that intermediate varieties, from existing in lesser numbers than the forms which they connect, will generally be beaten out and exterminated during the course of further modification and improvement. The main cause, however, of innumerable intermediate links not now occurring everywhere throughout nature depends on the very process of natural selection, through which new varieties continually take the places of and exterminate their parent-forms. But just in proportion as this process of extermination has acted on an enormous scale, so must the number of intermediate varieties, which have formerly existed on the earth, be truly enormous. Why then is not every geological formation and every stratum full of such intermediate links? Geology assuredly does not reveal any such finely graduated organic chain; and this, perhaps, is the most obvious and gravest objection which can be urged against my theory. The explanation lies, as I believe, in the extreme imperfection of the geological record. "

C Darwin "On the Origin of Species" 1859 chapter IX: "On the Imperfection of the Geological Record"

The utilisation of the longest ongoing experiment gives access to previous analysis and allows us to complement what has already been observed in these lines. The levels of single nucleotide polymorphisms [START_REF] Wielgoss | Mutation rate dynamics in a bacterial population reflect tension between adaptation and genetic load[END_REF] , changes in global regulator activity [START_REF] Cooper | Expression profiles reveal parallel evolution of epistatic interactions involving the CRP regulon in Escherichia coli[END_REF] or regulatory genes [START_REF] Plucain | Epistasis and allele specificity in the emergence of a stable polymorphism in Escherichia coli[END_REF] expression of cryptic metabolic pathways [START_REF] Blount | Genomic analysis of a key innovation in an experimental Escherichia coli population[END_REF]) have been extensively studied in the 12 populations in up to 40,000 generations. This provides a large understanding of evolved phenotypes and their beneficial effect to the cell. However, chromosomal rearrangements and movements of IS elements have been less studied [START_REF] Papadopoulos | Genomic evolution during a 10,000generation experiment with bacteria[END_REF][START_REF] Schneider | Long-term experimental evolution in Escherichia coli. IX. Characterization of insertion sequence-mediated mutations and rearrangements[END_REF] but their involvement in genetic innovation is crucial as illustrated by the tandem duplication involved in the cit+ phenotype [START_REF] Blount | Genomic analysis of a key innovation in an experimental Escherichia coli population[END_REF]. We only know of the IS dynamics up to 10,000 generations for two populations [START_REF] Papadopoulos | Genomic evolution during a 10,000generation experiment with bacteria[END_REF]) and the rearrangement profiles for the same two populations up to 2000 generations.

Rearrangements and Insertion Sequences are complimentary and both important actors in adaptation.

Part of this work was to extend the existing knowledge of rearrangements in the LTEE up to 40,000 generations in the twelve populations. In total we found 110 rearrangements, deletions, inversions and duplications all included in 1 strain issued from each of the twelve populations. Deletions were the most numerous, accounting for 75% of all rearrangements, inversions for 17% and duplications for 8%. IS elements are heavily implicated and account for 73% of the rearrangements. This could be expected as IS sequences are in multiple homologous copies distributed randomly throughout the genome and genomes with large numbers of IS copies can undergo more homologous recombination [START_REF] Rohmer | Comparison of Francisella tularensis genomes reveals evolutionary events associated with the emergence of human pathogenic strains[END_REF]) and previously described rearrangements in the twelve populations were all atributed to IS elements. Indeed, in all twelve populations IS elements have increased in copy numbers with a total of 209 IS signals detected by RFLP over 29 clones selected from the twelve populations at 40,000 generations. They were distributed randomly across the genome with no clear pattern, facilitating the potential for homologous recombination. However, even though they have a random insertion profile, parallel events occurred when IS sequences inserted in the rbs operon in all twelve populations [START_REF] Cooper | Mechanisms causing rapid and parallel losses of ribose catabolism in evolving populations of Escherichia coli B[END_REF]. In another evolution experiments on continuous cultures, IS elements were principal actors in rearrangements, 4% of the ancestor genome and delimited by IS1 element had been observed [START_REF] Maharjan | A case of adaptation through a mutation in a tandem duplication during experimental evolution in Escherichia coli[END_REF], and IS were involved in the disruption of rpoS, deletion involving mutY, yegS and yegR [START_REF] Gaffe | Insertion sequence-driven evolution of Escherichia coli in chemostats[END_REF]. Even though the reason for the presence and conservation of IS elements in bacterial genomes is under debate, it is recognise that IS elements play an essential role in the shaping of bacterial genomes by acting as the major source for rearrangements (Siguier et al 2014). This is especially true for the passage of a bacteria to an obligate intracellular pathogen where genome reduction is preceded by an explosion in the number of IS elements prior to massive reduction in chromosome size [START_REF] Bordenstein | Mobile DNA in obligate intracellular bacteria[END_REF]. As such in LTEE the increase in IS elements might favour an increase in rearrangement rates [START_REF] Papadopoulos | Genomic evolution during a 10,000generation experiment with bacteria[END_REF]. A previous study at 10,000 generations in the evolved population Ara-1 and Ara+1, shows that there are already large inversions present due to IS elements [START_REF] Papadopoulos | Genomic evolution during a 10,000generation experiment with bacteria[END_REF]). However, the present work indicates that IS elements are not the only participants in recombination;

other factors also participate in rearrangements in the evolving populations and in natural isolates [START_REF] Rau | Deletion and acquisition of genomic content during early stage adaptation of Pseudomonas aeruginosa to a human host environment[END_REF]). The second most common event cause was the deletion of prophage remnants. There were no homologous regions at either end of the majority of prophage deletions so it can be theorised that this type of recombination could be recA independent. Prophage elimination is a common feature where populations have evolved in environments where constitutively expressed prophage genes are not advantageous and they are often described as dispensable regions of DNA [START_REF] Varani | The role of prophage in plantpathogenic bacteria[END_REF].

Similar to IS elements, prophage sequences can play a role in homologous recombination [START_REF] Darling | Dynamics of genome rearrangement in bacterial populations[END_REF]) even though none of this type was observed in the LTEE .

DNA coding for rRNA is also a source for rearrangements. There are 7 rRNA operons in the ancestor spanning over 5000bp with a very high degree of homology [START_REF] Jeong | Genome sequences of Escherichia coli B strains REL606 and BL21 (DE3)[END_REF]). There were only four inversion involving rRNA sequences and even though there effect is not known. Rearrangements and IS elements participate in a major way in the populations adaptation to the experimental evolution as evidenced by their relatively high frequencies. Even though the exact effect of the majority of these events are not known, there is strong argument that they could be beneficial due to the high levels of parallelism observed between the different populations.

Parallelism in the Long Term evolution Experiment

The evolution of the 12 populations is characterized by a strong phenotypic parallelism:

fitness and cell volume increase [START_REF] Lenski | Dynamics of adaptation and diversification: a 10,000-generation experiment with bacterial populations[END_REF]Cooper and Lenski 2000;[START_REF] Lenski | Phenotypic and Genomic Evolution during a 20,000-Generation Experiment with the Bacterium Escherichia coli[END_REF][START_REF] Wiser | Long-term dynamics of adaptation in asexual populations[END_REF]. Globally the cells increase their ability to use glucose as a source of carbon which is the main carbon source in the evolution environment whereas the ability to use other sources of carbon not present in the evolution media such ribose or maltose is reduced (Cooper and Lenski 2000;[START_REF] Cooper | Mechanisms causing rapid and parallel losses of ribose catabolism in evolving populations of Escherichia coli B[END_REF][START_REF] Pelosi | Parallel changes in global protein profiles during longterm experimental evolution in Escherichia coli[END_REF]. Simultaneously, the expression of many genes is affected in parallel in the populations [START_REF] Cooper | Parallel changes in gene expression after 20,000 generations of evolution in Escherichiacoli[END_REF][START_REF] Pelosi | Parallel changes in global protein profiles during longterm experimental evolution in Escherichia coli[END_REF]) by modifications in the activity of global regulators such as CRP [START_REF] Cooper | Expression profiles reveal parallel evolution of epistatic interactions involving the CRP regulon in Escherichia coli[END_REF]. In ten populations, the chromosome superhelicity is increased [START_REF] Crozat | Parallel genetic and phenotypic evolution of DNA superhelicity in experimental populations of Escherichia coli[END_REF]) through major modifications to fis expression [START_REF] Crozat | Long-term experimental evolution in Escherichia coli. XII. DNA topology as a key target of selection[END_REF].

The optical mapping and the whole genome sequencing of clones from the 12 populations allowed us to observe a high level of parallelism in the chromosomal rearrangements. There were a total of nine regions where deletions were parallel in two or more populations, including one previously analysed deletion of the operon containing the genes necessary for the utilisation of ribose in all twelve populations [START_REF] Cooper | Mechanisms causing rapid and parallel losses of ribose catabolism in evolving populations of Escherichia coli B[END_REF]. This deletion occurred between an ancestral IS150 and newly inserted IS150 that inserted in the same operon in the twelve populations but at different locations [START_REF] Cooper | Mechanisms causing rapid and parallel losses of ribose catabolism in evolving populations of Escherichia coli B[END_REF]). It has become fixed in all twelve populations due to the fitness benefit associated with it. IS insertion signatures tended to be specific in each population and even between each strain within populations studied. This shows the largely stochastic method of IS dissemination. The fitness benefits of the other deleted regions are not known, however, since they mostly involve cryptic prophage remnants that do not encode for any vital genes, it can be supposed that their elimination will be beneficial. The prophage remnants are defective and do not code for any virulence genes however, λ-prophages such as DLP12 and Rac, encode proteins which might cooperate in the host physiology [START_REF] Canchaya | Prophage genomics[END_REF].

These prophages encode for the bor gene whose product is an outer membrane lipoprotein [START_REF] Barondess | bor gene of phage lambda, involved in serum resistance, encodes a widely conserved outer membrane lipoprotein[END_REF]. The protein was detected in various E. coli lysogens including many clinical isolates and plays a direct role in the pathogeny of E.coli by significantly and specifically increasing the survival of E. coli in animal sera [START_REF] Barondess | A bacterial virulence determinant encoded by lysogenic coliphage lambda[END_REF]. Therefore in the evolution media where constitutive expression of certain proteins is not essential for growth in minimal media the presence of prophages and their proteins would represent a cost to fitness to the cell. This could explain why the elimination of prophages is commonly observed. Another region that was deleted in 10 out of 12 populations was between two homologous genes manB and cpsG and also represented the loss of non-essential genes in the evolution environment. The strong parallelism between the populations suggests; even though there is no quantitative data, that all these deletions confer a fitness advantage in the evolution experiment.

A high level of parallelism was also detected for a duplication of a stretch of genome containing rpoS the sigma factor involved in stationary phase gene expression and stress response (Hengge-Aronis 1993). Our analysis indicates that the duplication occurred in 10 out of the 12 populations, involved various IS elements as IS1, IS150, IS186, depending of the population and six different sizes for these duplications have been noted. Heterology of duplications could even be observed within two populations. This could be due to duplications being bound to the stochastic profile of IS insertion. The complete parallel duplicated region comprised rpoS, nlpD, pcm, surE, truD, ispF, ispD, ygbE, ftsB, cysC, cysN, cysD and iap. Along with rpoS the genes nlpD, pcm and surE are all essential for stationary phase survival and might be the targets of the duplication as the populations spend most of their time in stationary phase during the evolution experiment. The other operons are involved in the pathway for the biosynthesis of isoprenoids (ispF and ispD) [START_REF] Campbell | Characterization of the depletion of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate synthase in Escherichia coli and Bacillus subtilis[END_REF] and for the sulphate activation pathway of ATP-sulphurylase (cysC, cysN and cysD) [START_REF] Leyh | The sulfate activation locus of Escherichia coli K12: cloning, genetic, and enzymatic characterization[END_REF]) and two genes (ftsB and iap) involved in cell divison [START_REF] Buddelmeijer | A complex of the Escherichia coli cell division proteins FtsL, FtsB and FtsQ forms independently of its localization to the septal region[END_REF]) and an alkaline phosphatase isozyme conversion protein, respectively. While there is no information about the contribution of each duplicate gene on the bacterial fitness, the most probable target for duplication remains the global regulator rpoS. It is also a target in other evolution experiments highlighting its importance [START_REF] Ferenci | The spread of a beneficial mutation in experimental bacterial populations: the influence of the environment and genotype on the fixation of rpoS mutations[END_REF]).

In the LTEE another duplication event gave rise to a clone capable of using citrate as a carbon source in the population Ara-3 [START_REF] Blount | Historical contingency and the evolution of a key innovation in an experimental population of Escherichia coli[END_REF], Blount, Barrick 2012 ).

The tandem duplication of a 2.9 kb DNA fragment containing citT encoding for a citrate/succinate antiport resulted in the gene being placed under the control of the aerobically expressed promoter of rnk. However, when the evolution experiment on precusor clones was replayed from a clone that had not yet evolved the duplication, out of the 270 attempts only 17 gave the Cit+ phenotype. They were not all due to a duplication and they included 6 results from a cryptic promoter located at the terminal part of a newly transposed IS3 element increased the expression of the citT gene [START_REF] Blount | Genomic analysis of a key innovation in an experimental Escherichia coli population[END_REF]. Duplications have been identified in other evolution experiments, however, due to their inherent instability their fitness effects are elusive [START_REF] Maharjan | A case of adaptation through a mutation in a tandem duplication during experimental evolution in Escherichia coli[END_REF]), but, similar to deletions, the level of parallelism suggests that it is beneficial. There were three regions of the genome that were affected by inversions between two or more populations. They varied in size from 364,762bp to 1,368,196bp and involved a large number of genes. In one shared inversion we noticed a stretch of 164,027bp that is inverted in eight of the twelve populations covering a large number of genes but also the dif region where the sister chromosomes are segregated after duplication [START_REF] Stouf | FtsK actively segregates sister chromosomes in Escherichia coli[END_REF]. Therefore gene orientation is maintained in these cases.

It is nearly impossible to know the effect of these large inversions as they occur in already well adapted populations where there are many other mutations that have large effects on cell fitness. It can be theorized that these inversions are beneficial to the populations since they occur in the majority of populations. Barrick et al. 2012) and the ability to grow on cell culture by product leading to two coexisting subpopulations in Ara-2 [START_REF] Plucain | Epistasis and allele specificity in the emergence of a stable polymorphism in Escherichia coli[END_REF]. We show that, based on IS -RFLP profiles, there might also be two co-evolving subpopulations in the population Ara-6.

Benefits to evolution of rearrangements and IS elements

This study identifies a large number of rearrangements and IS insertions. Unfortunately the fitness effects of many of these events are unknown due to their size and their complexity.

There has only been two rearrangement events that have been characterised, the ribose operon deletion in all twelve strains increased the fitness by 1 to 2% compared to the ancestor [START_REF] Cooper | Mechanisms causing rapid and parallel losses of ribose catabolism in evolving populations of Escherichia coli B[END_REF], the duplication leading to citrate utilization in Ara-3 gave the population a 1% advantage in relation to a clone without a duplication; however, more importantly this duplication was potentiating step for more mutations [START_REF] Blount | Historical contingency and the evolution of a key innovation in an experimental population of Escherichia coli[END_REF], Blount, Barrick et al. 2012). Changes on fitness brought by IS are even less studied, excepted for one clone in the Ara+1 population at 2000 generations where IS elements account for almost all its fitness increase compared to the ancestor. From the presented results, it is difficult to comment on specific rearrangements and IS movements, however when compared to other observations, it can be hypothesized that the majority of these events are beneficial, in particular in the case of parallel events. IS elements are thought to maintain themselves in the genome via two opposing mechanisms, either as parasites with a high rate of transposition and through dissemination via horizontal transfer or a beneficial actors that create enough beneficial mutations that they are maintained in the cell (Siguier, Gourbeyre et al. 2014). Using the LTEE we show that early IS insertions are beneficial to the cell and can be responsible for the majority of its fitness increase. Transposition then increase linearly in relation to the number of IS copies on the chromosome. We are able to use this data to propose a simple model for IS invasion. The model shows that if enough beneficial IS insertions occur then they are able to maintain themselves through parasitic behavior. This

shows that both previous theories of IS dissemination are essentially correct.

Rate of events and implication in natural isolates

The levels of rearrangements and IS changes, 2.3 x 10 -4 and 6.2 x 10 -4 per generation, respectively (all types added together) were within previously estimated levels for rearrangements 10 -3 ~10 -5 and IS 3.5x 10 -4 (in E.coli K12 batch cultures) [START_REF] Sousa | Rates of transposition in Escherichia coli[END_REF]. These rates are mostly inferred from experimental evolution experiments and are not entirely representative of what occurs in natural isolates. Compared to point mutations, descriptions of reorganization events in clinical or natural isolate of E. coli are relatively rare while IS movements are largely underreported. This suggests that either rearrangements are not beneficial in a complex environment or counter selected during the horizontal transfers maintaining the chromosomal integrity. A more trivial alternative might be that these rearrangements are not easily detected, as already report during experimental evolution [START_REF] Maharjan | A case of adaptation through a mutation in a tandem duplication during experimental evolution in Escherichia coli[END_REF]. IS movements have been largely described in relation to the passage of bacteria to endosymbionts, where the transposition rate has not been described, only that IS elements eventually account for up to 23% of the genome [START_REF] Gil | Massive presence of insertion sequences in the genome of SOPE, the primary endosymbiont of the rice weevil Sitophilus oryzae[END_REF]. In other conditions, IS changes have been largely understated. This could be due to difficulties in detection by sequencing or a difficulty in understanding their impact on the genome. To overcome these limitations new technologies have been developed and are now routinely used to investigate chromosomal changes in clinical isolates even though the rate of these events are not usually given as there is an unknown number of generations between the compared strains. The use of optical mapping on E. coli O157 :H7 natural isolates from a 2006 outbreak demonstrated that large chromosomal inversion (up to 1.6 Mbp) are observed in 2 out of the 11 populations analysed and eventually involved complex events resulting from sequential inversions (Iguchi terajima et al. 2006). Chromosomal rearrangements have been detected in clinical isolates of Samonella enterica [START_REF] Matthews | Chromosomal rearrangements in Salmonella enterica serovar Typhi strains isolated from asymptomatic human carriers[END_REF],

Bordetella pertussis, Neisseria meningitidis or N. gonorrhoeae where an inversion corresponding to more than one third of the chromosome had been found, [START_REF] Spencer-Smith | Sequence features contributing to chromosomal rearrangements in Neisseria gonorrhoeae[END_REF]). In P. aeruginosas large chromosomal inversions are induced by transposition and recombination between insertion sequences which in parallel with gene inactivation may improve the fitness of the bacteria through a trial and error process [START_REF] Rau | Deletion and acquisition of genomic content during early stage adaptation of Pseudomonas aeruginosa to a human host environment[END_REF]. In most of these cases IS elements are the source for the rearrangements.

Limitations of events.

In general there are more conditions that have been described limiting the scale of rearrangements but not of IS movements. The main limiting factor is the change in fitness brought by each event. There are limits on rearrangements due to the structure of the genome.

E. coli genome is organized in 4 macrodomains corresponding to structured segments of the chromosome and two inter macrodomain regions that correspond to unstructured segments of the chromosome. The overall function and structure of these macrodomains is still unclear [START_REF] Boccard | Spatial arrangement and macrodomain organization of bacterial chromosomes[END_REF]. It is generally thought that within these macrodomains rearrangements are possible but between macrodomains they are deleterious [START_REF] Esnault | Chromosome structuring limits genome plasticity in Escherichia coli[END_REF]. This means that certain inversions are not permitted and cannot be observed. All observed inversions in this study and in previous studies of E.coli fell within these boundaries.

Résumé

Les réarrangements d'ADN à grande échelle, tels que inversions, amplifications, duplications, délétions, insertions et transposition des éléments génétiques mobiles, sont des acteurs essentiels de l'évolution. Ils ont une forte incidence sur l'organisation et l'expression des chromosomes, ce qui affecte le phénotype des organismes. Toutefois, la dynamique de ces réarrangements au cours de l'évolution et leurs effets sur l'adaptation des organismes sont souvent inconnus.

Figure 1 Figure 2 .Figure 3 .Figure 4 .Figure 5 .Figure 6 .Figure 7 .Figure 8 .Figure 9 .

 123456789 Figure 1 Mechanisms by which recombinations via RecA can occur

Figure 10 .

 10 Figure 10. Divergence of the population Ara-2.

Figure 11 .

 11 Figure 11. Phylogeny pf Ara-3.

Figure 12 .

 12 Figure 12. Duplication of citrate transport genes in Ara-3

Figure 1 :

 1 Figure 1: Mechanisms by which recombinations via RecA can occur. The principal activity of RecA is to fill gaps in replication and to rescue replication fork collapse which has a secondary effect leading to recombination between the two DNA strands. A. Example of DNA repair mechanism of RecA. B. Example of a gap repair. Both cases require I: unravelling of coiled DNA. II: binding of RecA machinery. III: Strand exchange due to homologous sequences. IV: resolution of Holliday Junction and V: restart of replication fork (adapted from Michel 2007).

Figure 2 :

 2 Figure 2: The three major types of homologous rearrangement. The three major chromosomal rearrangements, deletions, duplications and inversions are represented here. Thick black lines indicate the chromosome, boxes indicate homologous sequences and the arrows above them indicate the direction of their homology. Letters indicate various chromosomal locations.

Figure 3 :

 3 Figure 3: Explanation of Whole Genome Mapping TM (i.e.optical mapping). Methodology used by OpGen© to construct optical maps from purified DNA. A. DNA molecules are stretched and fixed to a glass plate by charge. B. Fixed DNA is washed with a restriction enzyme. C. Restriction profiles and fragment lengths are taken. D. Multiple fragments are used to create a whole genome profile. E. Genome profiles are compared to an in silico reference (adapted from Shukla, Kislow et al. 2009)

Figure 4 :

 4 Figure 4: Stability of the duplication and phenotypes of the different derived genotypes. The proportion of clones carrying duplications or derived copies in different conditions after three days of culture. HD stands for clones with the duplication WT and DO stand for clones that have either one wild type copy or one evolved copy of the duplication. A. In the evolution conditions (chemostat). B. In another condition (LB 24hr batch cultures) (adapted from Maharjan, Gaffe et al. 2013).

Figure 5 :

 5 Figure 5: Organization of a typical IS. The IS is represented as an open box in which the terminal IRs are shown as blue arrows labelled IRL (left inverted repeat) and IRR (right inverted repeat). A single open reading frame encoding the transposase is indicated as a green arrow. XYZ enclosed in a pointed box flanking the IS represents short DR (Direct Repeat) sequences generated in the target DNA as a consequence of insertion.

  ; (2) the length and sequence of the short imperfect terminal inverted repeat sequences (IRs) varying from 10-40bp carried by many ISs at their ends; (3) the length and sequence of the short flanking direct target DNA repeats (DRs) often generated on insertion up to 10bp; (4) the organisation of their open reading frames or (5) the target sequences into which they insert (Fig 5)

  IS4 and IS5), named after the conservation of two Asp and a Glu at the active site. It can be encoded by one or two ORF and are closely related to the retroviral integrase catalytic core (IN) by the spacing of the DDE triad and by the appearance of additional conserved residues. The Holliday junction resolvase RvuC is also related. A structurally related transposase type only found in the IS family IS110 is DEDD (three Asp and a Glu); however, they do not create inverted repeats nor direct repeats upon insertion as observed with DDE transposase. The second family encodes for a tyrosine transposase (or HUH transposase) (Ronning and Guynet 2005), largely present in the family IS91, IS2000 and IS605. The transposase contains no inverted repeats nor does it cause a direct repeat upon insertion. Transposition occurs through the formation of a hairpin secondary Structures coded for by subterminal sequences. The third major family of transposase are serine transposases carried by the families IS607. They are closely related to serine resolves carried by Tn3 elements. Interestingly they are the only bacterial transposase family that are present in several eukaryote genomes, but are mostly likely present due a capture of a longer bacterial DNA through infection rather than capture via transposition (Gilbert and Cordaux 2013).

Figure 6 :

 6 Figure 6: Circular copy-paste IS transposition mechanism common to the IS3 family. A. Representation of a dsDNA (blue line) containing a IS element (green line) B. A nick is generated at one 3' end. The resulting 3' OH attacks the same strand immediately outside the transposon (dotted line with arrow). C. Replication is primed from the nick at the transposon junction resolves this intermediate into D. an excised transposon circle. E. A second nick occurs at the ends of the transposase to create a linear product. F. The transposases then inserts the transposon between two staggered nicks in the target which is repaired by host enzymes

Figure 8 :

 8 Figure 8: Average fitness distribution of the 12 evolving populations. Hyperbolic (red) and power-law (blue) models fit to the set of mean fitness values (black symbols) from all 12 populations (adapted from Wiser, Ribeck et al. 2013).

Figure 9 :

 9 Figure 9: Insertion of an IS150 element into nadR in population Ara + 1, and a subsequent inversion involving that new element. Example of an inversion detected in the evolving populations. A. A new IS150 copy inserted into the gene nadR. B. subsequent rearrangement between newly inserted IS and an ancestral IS located in the intergenic region of hokX-sokX (adapted from Schneider, Duperchy et al. 2000).

  the capabilities for S to maintain itself in early emergence(Plucain, Hindre et al. 2014) (Fig 10).

Figure 10 :

 10 Figure 10: Divergence of the population Ara-2. The population Ara-2 diverged into two co-existing ecotypes after 6,000 generations. Three mutations, in arcA, spoT, and gntR have been shown to cause the S phenotype. Evolved genotypes are shown in circles (adapted from Plucain, Hindre et al. 2014).

Figure 11 :

 11 Figure 11: Phylogeny pf Ara-3. Symbols at branch tips mark 29 sequenced clones; labels are shown for clones mentioned in main text and figures. Shaded areas and coloured symbols identify major clades. Fractions above the tree show the number of clones belonging to the clade that yielded Cit + mutants during replay experiments (numerator) and the corresponding total used in those experiments (denominator). Inset shows number of mutations relative to the ancestor. The solid line is the least-squares linear regression of mutations in non-mutator genomes; the dashed line is the corresponding regression for mutator genomes. (adapted from Blount, Z. D., J. E. Barrick, et al. (2012))

Figure 12 :

 12 Figure 12: Duplication of citrate transport genes in Ara-3. Duplication bordered by IS3 explains the evolution of citrate utilisation in the population Ara-3.The genes citT and rna and a part of citG are duplicated behind a strong promoter of rnk. a: the ancestral genome b:Evolved genome (adapted from Blount, Z. D., J. E.Barrick, et al. 2012).

FIG 1

 1 FIG 1Large-scale chromosomal rearrangements in evolved clones sampled after 40,000

FIG 2 FIG 3 FIG 4 FIG 1

 2341 FIG 2 Successive inversions in population Ara+1. (A) Optical map of the genome of the

FIG 2 FIG 3

 23 FIG 2 Successive inversions in population Ara+1. (A) Optical map of the genome of the

5 .

 5 In relation with the above point 1, the authors should consider to mention (in the discussion) the paper from Moore JM. et al. 2012. Ann N Y Acad Sci. 2012 Sep;1267:103-9. doi: 10.1111/j.1749-6632.2012.06587.x. Gross chromosomal rearrangement mediated by DNA replication in stressed cells: evidence from Escherichia coli". They underline the possible effect of RecA, which is not mentioned in the manuscript under revision. 6. Also in relation with the above point 1, the authors might consider the paper from Lin D. et al., 2011. Global chromosomal structural instability in a subpopulation of starving journal.pgen.1002223. A sentence in the discussion about the relation between stress

  mbio.msubmit.net/cgibin/main.plex?el=A2FE7chE4A6GqE3I3A9ftdq059VOGUEiNXKL8iZu8udgZ. You should include point-by-point responses to the issues raised during review in a file labeled "Response to Reviewer Comments."

  Darwinian selection drives the dynamics of IS elements during bacterial long term evolution State: To be submitted to PNAS Significance Statement

Fitness

  effect of early IS150 dynamics in Ara+1. No changes in either IS150 sequence or transposition frequency can explain the initial strong copy number increase. The genome sequence of the 2000-generation clone 1158C, which was shown to be on the line of descent of population Ara+1, revealed already the presence of three new IS150 insertions and two IS150-related deletions. The three new insertions accounted for one third of all mutations (Table

  and IS dynamics. The code of the model that we developed has been deposited in the Dryad Digital Repository (doi: deposition pending acceptance). The model includes an initial simulated population of 10 7 digital organisms with each individual having a fitness of 1. The population experienced seasonal cycles consisting in a 100-fold expansion before going through a bottleneck to be reduced back to 10 7 . During each cycle, the fitness (F) of each individual can be altered by substitution of a mutation among a pool of 20,000

Fig. 1 .

 1 Fig. 1. Proportion of IS-related changes relative to total mutations after 40,000

Fig. 2 .

 2 Fig. 2. Dynamics of IS-related changes and of their contribution to total mutations in

Fig. 3 .

 3 Fig. 3. Recombination and transposition frequencies of IS150 in the ancestor and five

Fig. 4 .

 4 Fig. 4. Simulation of IS dynamics and contribution relative to point mutations. Boxplots

Fig. 1 .

 1 Fig. 1. Proportion of IS-related changes relative to total mutations after 40,000 generations.

Fig. 2 .

 2 Fig. 2. Dynamics of IS-related changes and of their contribution to total mutations in

Fig. S2 .

 S2 Fig. S2. Phylogenies for 28 evolved clones sampled from the 12 populations of E. coli at

Fig. S5 .

 S5 Fig. S5. Dynamics of IS elements in three of 100 replicate populations (μ m = 2 x 10 -5 , μ IS =

  ABSTRACT Large-scale DNA rearrangements may be important drivers of evolution because they can alter chromosome organization and gene expression in ways not possible through point mutations. In a long-term evolution experiment, twelve E. coli populations have been propagated in a glucose-limited environment for over 25 years. We used Whole

	Genome Mapping TM (i.e. optical mapping) combined with genome sequencing and PCR
	analysis to identify the large-scale chromosomal rearrangements in clones from each
	population after 40,000 generations. A total of 110 independent rearrangement events were
	detected, including 82 deletions, 19 inversions and 9 duplications, with different lineages

a,b* Univ. Grenoble Alpes, Laboratoire Adaptation et Pathogénie des Microorganismes (LAPM), F-38000 Grenoble, France a ; Centre National de la Recherche Scientifique (CNRS), LAPM, F-38000 Grenoble, France b ; Department of Molecular Biosciences, Institute for Cellular and Molecular Biology, Center for Systems and Synthetic Biology, The University of Texas at Austin, Austin, TX 78712 c ; IAME, UMR 1137, INSERM, F-75018 Paris, France d ; IAME, UMR 1137, Université Paris Diderot, Sorbonne Paris Cité, F-75018 Paris, France e ; OpGen, Inc., Gaithersburg, MD 20878 f ; Direction des Sciences du Vivant, Commissariat à l'Energie Atomique et aux Energies Alternatives (CEA), Institut de Génomique, Genoscope & CNRS-UMR8030, Evry, France g ; Laboratoire d'Analyses Bioinformatiques en Génomique et Métabolisme (LABGeM), Evry, France h ; Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824, USA i ; BEACON Center for the Study of Evolution in Action, Michigan State University, East Lansing, MI 48824, USA j Running Head: Rearrangements during experimental evolution † Present address: DNASTAR, Inc., Madison, WI 53705, USA.

TABLE S1

 S1 Rearrangements detected in 40,000-generation clones sampled from each of the 12 populations of the LTEE

	Population Ara+1
	Type	Start a	End a	Size (bp) Mechanism b	Genes altered c	Parallelism d
	Inversion 1 e 4,615,673	2,774,435	2,788,574 RRE-IS150	nadR-insK-3, 2661 genes	I1
	Inversion 2 e 490,480	2,877,312	607,496	RRE-IS150	nadR-ybbN and cysH-kduD, 549 genes
	Inversion 3 e 353,643	2,877,312	470,659	RRE-IS150	nadR-yaiO and cysH-kduD, 418 genes
	Inversion 4 e 146,102	2,877,312	263,118	RRE-IS150	nadR-yadG and cysH-kduD, 224 genes
	Deletion	547,701	556,319	8618	RRE-IS1	DLP12-like locus: ybcR, ybcS, ybcT, ybcU,
						ECB_00510, nohB, ECB_00512, ECB_00513
	Deletion	590,445	601,096	10,651	RRE-IS150	hokE, IS 186, entD, fepA, fes, ybdZ, entF, fepE
	Deletion	1,353,847	1,354,425	578	RRE-IS150	sapA
	Deletion	1,609,176	1,615,468	6292	RRE-IS3
						ECB_01523
	Deletion	2,034,326	2,053,851	19,525	RRE-IS1

Qin-like locus: ybcW, ynsB, ynfN, ECB_01516, cspI, ydfP, ydfQ-2, ydfR, essQ, ECB_01522,

  , alsB

	Deletion	547,701	555,877	8176	RRE-IS1	DLP12-like locus: ybcR, ybcS, ybcT, ybcU,
						ECB_00510,nohB, ECB_00512, ECB_00513
	Inversion	634,745	2,128,599	1,493,854 RRE-IS1	ycjW-gatA, 1455 genes	I1
	Deletion	2,031,703	2,054,996	23,293	RRE-manB-
	Deletion of				RRE-IS1
	duplication
	junction
	Population Ara+6
	Deletion	547,701	556,319	8618	RRE-IS1	DLP12-like locus: ybcR, ybcS, ybcT, ybcU,
						ECB_00510, nohB, ECB_00512, ECB_00513
	Deletion	1,433,349	1,442,639	9290	RRE-IS3
						ECB_01523
	Deletion	1,974,042	1,976,274	2232	RRE-IS1	yedU, yedV, yedW
	Deletion	2,100,286	2,122,432	22,146	Unknown	Prophage 2 locus: ogrK-ECB_02012, 26 genes
	Deletion	3,024,713	3,025,120	407	RRE-IS1	ECB_02825
	Deletion	3,775,351	3,796,244	20,893	Unknown	selC, ECB_03516, ECB_03517, ECB_03518,
						ECB_03519,	ECB_03520,	ECB_03521,
						ECB_03522,	ECB_03523,	ECB_03524,
						ECB_03525,	ECB_03526,	ECB_03527,
						ECB_03528,	ECB_03530,	ECB_03531,
						ECB_03532, ECB_03533, ECB_03534, ykgN,
						insI, ECB_03537, ECB_03538, ECB_03539,
						ECB_03540
	Deletion	3,894,996	3,900,156	5160	RRE-IS150	rbsD, rbsA, rbsC, rbsB, rbsK, rbsR
	Population Ara-1

hslJ, ldhA, ydbH, ynbE, ydbL, feaR, feaB,

tynA Deletion 1,609,176 1,615,468 6292 RRE-IS3 Qin-like locus: ybcW, gnsB, ynfN, ECB_01516, cspI, ydfP, ydfQ-2, ydfR, essQ, ECB_01522,

  ybdN

	Inversion	1,443,893	1,607,920	164,027	RRE-IS3	ybdC-ECB_01510, 148 genes	Sub-region
							1.1
	Deletion	1,609,176	1,615,468	6292	RRE-IS3
						ECB_01523
	Deletion	2,031,432	2,054,725	23,293	RRE-manB-

Qin-like locus: ybcW, ynsB, ynfN, ECB_01516, cspI, ydfP, ydfQ-2, ydfR, essQ,ECB_01522,

TABLE S2

 S2 Clones from the LTEE used in this study

	Population	Generation	Clone name
	Ancestor	0	REL606
	Ara+1	2000	REL1158A
	Ara+1	2000	REL1158B
	Ara+1	2000	REL1158C
	Ara+1	15,000	REL7183A
	Ara+1	15,000	REL7183B
	Ara+1	15,000	REL7183C
	Ara+1	20,000	REL9282A
	Ara+1	20,000	REL9282B
	Ara+1	20,000	REL9282C
	Ara+1	25,000	REL10241
	Ara+1	30,000	REL10450
	Ara+1	30,000	REL10451
	Ara+1	30,000	REL10452
	Ara+1	35,000	REL10796
	Ara+1	35,000	REL10797
	Ara+1	35,000	REL10798
	Ara+1 a	40,000	REL11008
	Ara+1	40,000	REL11009
	Ara+1	40,000	REL11010
	Ara+2 a	40,000	REL10950
	Ara+3 a	40,000	REL10953
	Ara+4 a	40,000	REL10956
	Ara+5 a	40,000	REL10982
	Ara+6 a	40,000	REL10985
	Ara-1 a	2000	REL1164A
	Ara-1 a	5000	REL2179A
	Ara-1 a	10,000	REL4536A
	Ara-1 a	15,000	REL7177A
	Ara-1 a	20,000	REL8593A
	Ara-1 a	30,000	REL10391
	Ara-1 a	40,000	REL10938
	Ara-1 a	50,000	REL11330
	Ara-2 a	40,000	REL11041
	Ara-3 a	40,000	REL10979
	Ara-4 a	40,000	REL10944
	Ara-5 a	40,000	REL10947
	Ara-6 a	40,000	REL11005

a Clones used for optical mapping.

TABLE S3

 S3 Primers used in this study

	Inversion 3 -Left +1Inv3LR	cctgagtttattggtcgtct
	Rearrangement a Ara+1 Inversion 4 -Right +1Inv3RR Name +1Inv4RF	Sequence (5' to 3') agagttcgcccggctcaaga tgcagtacgcaccagcactt	Expected PCR product size (bp) 2736
	Ara+1 Deletion 1 Ara+1 Inversion 4 -Left +1Inv2RR +1Del1F +1Del1R +1Inv4LF	ccttttgactgaaggtaagca tcaacagacacactacccgt ctgcttcttccaggtagtgt tggaatgcggtcgaggattt	2858 2214
	Ara+1 Deletion 2 Ara+2 Deletion 1	+1Del2F +1Del2R +2Del1F +2Del1R	ttaaccacctgcatttccag cagttcgtcgattttcgcct ccttttgactgaaggtaagca gcagtattgcgaccagatgg	2175 2195
	Ara+1 Deletion 3 Ara+2 Deletion 2	+1Del3F +1Del3R +2Del2F +2Del2R	atccccacggggatgccaacaa 2456 gtcctgggatgaacgtcgcct ttcgtaagcgagaacagcct 2442 taacatcaccctggatgtgc
	Ara+1 Deletion 4 Ara+2 Deletion 3	+1Del4F +1Del4R +2Del3F +2Del3R	gagccagatgggttttccct gtgatgaagccagaaaggca gcctgaccaaaatgggcgt tagccggaacctgtgggagca	3044 2261
	Ara+1 Deletion 5 Ara+2 Deletion 4	+1Del5F +1Del5R +2Del4F +2Del4R	accggaataagaggtgagct gagcttccgtaatcagccgtgg tatcaacggacctccacgga tcagcgttacaaggcttgga	2678 2749
	Ara+1 Deletion 6 Ara+2 Deletion 5	+1Del6F +1Del6R +2Del5F +2Del5R	gtctatcgctgacttgcgga gcctgtaaagccggtgacat aactctgcgcaccgaagacg agccatcggctttgaactgg	4129 3721
	Ara+1 Deletion 7 Ara+2 Deletion 6	+1Del7F +1Del7R +2Del6F +2Del6R	ttctcttgcgtgactgcctt ccggatattcacaatgtggcg ggggagaaagatgcacagtg taggaaacctcaatcggtca	3561 2440
	Ara+1 Deletion 8 Ara+2 Inversion 1 -Right +2-Inv1RR gttgcccgatcagaaaacgct +1Del8F aactctgcgcaccgaagacg +1Del8R tgcaaaatcgatggttaccca +2-Inv1RF attttgcgtcgtaagcgggagc 960 3592
	Ara+1 Inversion 1 -Right +1Inv1RR +1Inv1RF Ara+2 +2-Inv1LF accggaataagaggtgagct gcggtatgaccaaagggtat gcgggaaatagctggcatgac Inversion 1 -Left +2-Inv1LR acagcaaaaagacgaggtgg	2168 1053
	Ara+1 Inversion 1 -Left +1Inv1LR +1Inv1LF Ara+2 +2-Inv2RF tcaaacatcacccgaagatg taccgaataccacaccaatg acggcaaactgtgggaacagg Inversion 2 -Right +2-Inv2RR aaacgtctggaagaacgtgg	2099 2438
	Ara+1 Inversion 2 -Right +1Inv2RR +1Inv2RF Ara+2 +2-Inv2LF gtagacagcagctccacacc ccgctttacgttgggaccgg tggaatgcggtcgaggattt Inversion 2 -Left +2-Inv2LR tgagcactgcaaagtacgct	1660 2297
	a
	Ara+1 Inversion 2 -Left +1Inv2LR +1Inv2LF	aggctttccaggatcggggt gttatgggcgtgacgcgatt	1696
	Ara+1 Inversion 3 -Right +1Inv3RR +1Inv2RR	tggaatgcggtcgaggattt tgcagtacgcaccagcactt	3379
	Ara+1	+1Inv2RF	ccgctttacgttgggaccgg	4239

TABLE S4

 S4 Parallel rearrangements in the LTEE

	Region a	Population Start b	End b	Mechanism c	Genes affected in parallel d
	547,701-550,351:
		Ara+1	547,701	556,319	RRE-IS1	DLP12-like locus: ybcR, ybcS, ybcT, ybcU,
		Ara+2	547,701	588,493	RRE-IS1	ECB_00510,nohB, ECB_00512, ECB_00513
		Ara+3	547,701	588,493	RRE-IS1
		Ara+4	547,701	559,507	RRE-IS1
		Ara+6	547,701	556,319	RRE-IS1
		Ara-1	547,701	555,877	RRE-IS1
		Ara-3	547,701	550,351	RRE-IS1
		Ara-4	547,701	619,884	RRE-IS1
		Ara-5	547,701	558,574	RRE-IS1
		Ara-6	547,701	589,555	RRE-IS1
	590,472-601,096:
		Ara+1	590,445	601,096	RRE-IS150	hokE, insL-3, entD, fepA, fes, ybdZ, entF, fepE
		Ara-2	590,472	619,116	RRE-IS1
		Ara-4	547,701	619,884	RRE-IS1

1,609,176-1,615,468:

	Ara+1	1,609,176 1,615,468	RRE-IS3	Qin-like locus: ybcW, ynsB, ynfN, ECB_01516, cspI,
	Ara+6	1,609,176 1,615,468	RRE-IS3	ydfP, ydfQ-2, ydfR, essQ,ECB_01522, ECB_01523
	Ara-2	1,609,176 1,615,468	RRE-IS3
	Ara-3	1,609,176 1,615,468	RRE-IS3
	Ara-4	1,609,176 1,615,468	RRE-IS3
	Ara-5	1,609,176 1,615,468	RRE-IS3

Table S1 . E. coli strains used in this study

 S1

	10979	Ara-3	40,000	SRA026813 ‡
	Strain 10988	Population Generation Accession Ara+2 40,000 Ara-3 40,000 SRA026813 ‡
		Ara+2	40,000
	REL606		Ancestor
	10944	Ara-4	40,000
	768A	Ara+1	500
	10945	Ara+3 Ara-4	40,000 40,000
	768B	Ara+1	500
		Ara+3	40,000
	958A	Ara+1	1000
	10947	Ara-5	40,000
	958B	Ara+1	1000
	10948	Ara+4 Ara-5	40,000 40,000
	1062A	Ara+1	1500
	10949	Ara+4 Ara-5	40,000 40,000
	1062B	Ara+1	1500
	1158A	Ara+1	2000
	11005	Ara+5 Ara-6	40,000 40,000
	1158C	Ara+1	2000
	11006	Ara+5 Ara-6	40,000 40,000
	2173A	Ara+1	5000
	2173B All of the strains are evolved clones derived from the REL606 strain (35) and sampled from Ara+1 5000
	4530A the 12 populations of the long-term evolution experiment. Ara+1 10,000 Ara+6 40,000
		Ara+6	40,000
	4530B	Ara+1	10,000
	7183A	Ara+1	15,000
		Ara-1	40,000	SRS007219 †
	7183B	Ara+1	15,000
		Ara-1	40,000	ERS068526 †
	9282A	Ara+1	20,000
		Ara-1	40,000	ERS068529 †
	9282B	Ara+1	20,000
	10450 1. Blount ZD, Barrick JE, Davidson CJ, Lenski RE (2012) Genomic analysis of a key Ara+1 30,000
	10451 innovation in an experimental Escherichia coli population. Nature 489(7417):513-518. Ara+1 30,000 Ara-2 40,000
		Ara-2	40,000
	11008	Ara+1	40,000
		Ara-2	40,000
	11009	Ara+1	40,000
	11010 * Ara+1 Ara-2	40,000 40,000
				153 154

*

This particular 40,000-generation clone was used only to measure the frequency of IS150 transposition whereas all other clones from the same time point were used in all the analyses. † These genome sequences are from (4). ‡ These genome sequences are from

(ref. 1)

.

Table S3 . Number of copies of the five active IS elements in the ancestor and of IS- related changes in each of the 28 evolved clones sampled at 40,000 generations.

 S3 Data are from the IS fingerprints. For the ancestor, the copy number is given. For the evolved clones, the number of changes in IS-hybridizing bands is given, including therefore the loss and gain of IS copies. The data for IS2 and IS30 (1 copy each in the ancestor) are not shown since no change was detected.

		10988	11	4	0	23	8
	Ara-4	10944	8	3	0	5	1
	Population Clone 10945	IS1 6	IS3 3	IS4 IS150 IS186 0 5 1
	Ara-5	10947	6	3	0	14	6
		REL606	24	5	2	5	5
		10948	5	3	1	15	6
	Ara+1	11008	11	3	0	29	7
		10949	6	3	1	15	6
		11009	11	3	0	29	7
	Ara-6	11005	8	1	0	12	6
	Ara+2	10950	9	0	0	8	7
		11006	6	1	0	10	2
		10951	9	0	0	8	5
	Ara+3	10953	2	0	2	4	4
		10954	2	0	1	4	4
	Ara+4	10956	5	0	3	3	0
		10957	6	0	2	3	0
	Ara+5	10982	6	0	0	14	3
		10983	8	0	0	14	3
	Ara+6	10985	1	3	0	5	2
		10986	1	3	0	6	1
	Ara-1	10938	12	0	0	7	5
		10939	13	3	0	8	4
		10940	13	3	0	8	4
	Ara-2	11035	7	4	0	8	1
		10941	8	4	0	8	2
		11036	8	3	0	11	6
		11214	7	3	0	11	4
	Ara-3	10979	10	4	0	17	8
					156

Table S4 . Primers used in this study to PCR-amplify 19 new IS150 copies in evolved clones

 S4 If the insertion of a new IS150 copy occurred within a gene, its name is given. If the insertion occurred in an intergenic region, the name of the upstream gene is given together with the distance in basepairs, in brackets, between the last base of its stop codon and the IS150 insertion.

	IS150-15-R tttgctctatctggggagta	1,859,061
	IS150-16-F ggcaacatttgtagaccaga	2,019,729	yeeZ(196)	2,019,802
	Primer IS150-16-R ggcctggagtgactacgtta Sequence 5'-3' IS150-3-F tgatgttgaactggaagtcg IS150-19-F gccctaatacgacaaaagcc	Position * 2,019,849 335,981 2,283,403	Gene † lacZ yfaA(21)	Position of IS insertion * 336,055 2,283,472
	IS150-3-R IS150-19-R tgaaatcgctccggaagtgg tgttttgaccgctgggatct	336,100 2,283,522
	IS150-5-F IS150-20-F gtttggtatgcgagtgggta ccgattatcctctggcgtcg	601,005 2,397,339	fepE yfcT	601,096 2,397,529
	IS150-5-R IS150-20-R cgaacatgcaacgtaccgga gctgatctctttcatcctgc	601,126 2,397,641
	IS150-6-F IS150-21-F gcggtaatcacttcccataa cccctgggtgaatcaaatag	651,527 2,591,569	ybeB(177) hcaC	651,703 2,591,547
	IS150-6-R IS150-21-R taaaactcgccgccaacgtt gccagacacgactttgtaga	651,777 2,591,594
	IS150-7-F IS150-22-F cgtaccggatcgtcttgatt cccgctgccgttcatcctct	711,881 2,655,684	rhsC yfiH	711,955 2,655,779
	IS150-7-R IS150-22-R ctggcggatatttatctgct gcggatatccagattcgctg	712,001 2,655,853
	IS150-8-F IS150-23-F tggaatgcggtcgaggattt ggataacggttcgctggttg	896,391 2,877,238	ECB_00838 kduD	896,463 2,877,312
	IS150-8-R IS150-23-R gttatgggcgtgacgcgatt ttcccgtaacgccgggttat	896,546 2,877,368
	IS150-10-F ctttcaggtcacgttcgatg IS150-26-F gggtataaggctgtagcgcc	969,768 3,493,736	pflB ECB_03279	969,836 3,493,822
	IS150-10-R accaacgctcaggaagctat IS150-26-R ccagtaacgccgtgcctaat	969,881 3,493,884
	IS150-11-F attttcgcccggtctacgac IS150-28-F gctggtaggtacaacagctgc	1,101,953 4,415,599	phoH cycA	1,102,032 4,415,710
	IS150-11-R cacattctgcgcctcgtcaa IS150-28-R gcagtaccagcgaaagggct	1,102,097 4,415,886
	IS150-12-F caagtatgcatcgctgatgg	1,376,305	ycjT	1,376,381
	IS150-12-R accagttcgttgatttcacc	1,376,429
	IS150-13-F atggcgggtaagaggctaag	1,462,199	hokB(313)	1,462,266
	IS150-13-R ggtgcctgagactttctgtt	1,462,306
	IS150-14-F tagcctgcttctccttgcct	1,519,763	hokD(544)	1,519,832
	IS150-14-R tgaacaccaacggcagatag	1,519,885
	IS150-15-F atggctttcggattagtcag	1,858,946	yeaS	1,859,000

*

Positions are given according to the ancestral genome sequence

(35)

. †

Table S5 . Mutations detected in two clones sampled from population Ara+1 at 2000 generations

 S5

	Population Clone	Position *	Mutation Type †		Affected Gene ‡ Effect
	Ara+1	REL1158A 70,867	NS-SNP	T->C	araA	D92G
	Ara+1	REL1158A 1,236,764 IS insertion	IS150	ycgB
	Ara+1	REL1158A 1,406,224 SNP	G->A	ydaN(254)
	Ara+1	REL1158A 1,462,266 IS insertion	IS150	hokB(313)
	Ara+1	REL1158A 1,733,865 NS-SNP	G->T	pykF	A301S
	Ara+1	REL1158A 2,847,052 NS-SNP	A->G	recD	V10A
	Ara+1	REL1158A 3,251,604 NS-SNP	T->G	nusA	Y386S
	Ara+1	REL1158A 3,761,696 NS-SNP	C->A	spoT	H314N
	Ara+1	REL1158A 3,894,998 IS150-mediated	6133 bp rbs operon
				deletion
	Ara+1	REL1158A 4,615,673 IS insertion	IS150	nadR
	Ara+1	REL1158C 70,867	NS-SNP	T->C	araA	D92G
	Ara+1	REL1158C 651,703	IS insertion	IS150	ybeB(177)
	Ara+1	REL1158C 664,687	IS150-mediated	1444 bp insJ, insK
				deletion
	Ara+1	REL1158C 1,462,266 IS insertion	IS150	hokB(313)
	Ara+1	REL1158C 1,733,865 NS-SNP	G->T	pykF	A301S
	Ara+1	REL1158C 2,847,052 NS-SNP	A->G	recD	V10A
	Ara+1	REL1158C 3,102,720 Insertion	1 bp	mqsR(201)
	Ara+1	REL1158C 3,440,702 NS-SNP	C->A	yhfZ	A109S
	Ara+1	REL1158C 3,894,998 IS150-mediated	6133 bp rbs operon
				deletion
	Ara+1	REL1158C 4,615,673 IS insertion	IS150	nadR

*

All positions are given according to the ancestral genome sequence

(35)

. † SNP : single-nucleotide polymorphism ; NS : non-synonymous. ‡ For intergenic mutations, the upstream gene is given together with, in brackets, the distance in basepairs from the last base of the stop codon to the mutation.

  Nous avons abordé ces questions en utilisant la plus longue expérience d'évolution en cours. A partir d'un ancêtre commun d'Escherichia coli, douze populations indépendantes sont cultivées dans un milieu limité en glucose depuis plus de 60 000 générations, soit 26 ans. La plupart des études antérieures ont porté sur les mutations ponctuelles et les petites insertions et délétions (InDels). En utilisant des clones isolés au cours du temps dans ces 12 populations, nous avons caractérisé les réarrangements d'ADN à grande échelle à la fois par l'analyse des séquences de génomes et par cartographie optique. A 40 000 générations, nous avons identifié 110 réarrangements parmi lesquelles 82 délétions, 19 inversions et 9 duplications. Plusieurs régions du chromosome ont été touchées à plusieurs reprises par le même type de réarrangements dans des populations indépendantes. Dans une des populations au moins, les réarrangements se sont produits au début de l'expérience d'évolution, au moment où l'augmentation de la valeur sélective est la plus élevée. Par conséquent, certains de ces réarrangements pourraient être bénéfiques dans ces conditions. Même dans le cas contraire, nous avons montré que ces réarrangements affectaient fortement la structure du chromosome au cours de l'expérience d'évolution.Au niveau moléculaire, nous avons montré que ~ 70% des réarrangements se produisent par recombinaison entre séquences d'insertion (IS), ce qui illustre l'importance de ces dernières dans la plasticité du génome. Nous avons donc caractérisé la distribution et la dynamique de ces petits éléments génétiques mobiles dans l'ensemble des 12 populations. Nous avons montré que les éléments IS ont fortement contribué à l'ensemble des mutations après 40 000 générations. Dans une population, les IS représentent même la moitié des mutations, et un des types d'IS, IS150, présente une forte prolifération avec 6 fois plus de copies à 40 000 générations, intervenant dans la plupart des réarrangements détectés dans cette population. Nous avons montré une forte dynamique temporelle d'IS150, avec une forte expansion suivie d'une domestication par l'hôte. En testant trois scenarii évolutifs, nous avons démontré que l'expansion d'IS150 était liée à une forte augmentation de la valeur sélective conférée par les événements initiaux de transposition ayant eu lieu avant 2000 générations. Plus tard, entre 20 000 et 40 000 générations, nous avons mesuré une diminution de la fréquence de transposition, probablement en raison d'une régulation négative de la transposition imposée par l'hôte. Enfin, et pour la première fois, nous avons développé un modèle d'évolution de la dynamique des IS, qui confirme que leur expansion est liée à un nombre seuil d'insertions bénéfiques initiales. Ces résultats montrent que les réarrangements chromosomiques à grande échelle et les éléments IS ont joué un rôle actif dans la trajectoire évolutive au cours de 40 000 générations d'évolution bactérienne.

short DNA reads cannot identify all large-scale rearrangements. Therefore, deciphering changes in the overall organization of genomes requires the use of alternative methods, such as optical mapping. We analyzed the longest-running microbial evolution experiment (more than 25 years of evolution in the laboratory) by optical mapping, genome sequencing, and PCR analyses. We found multiple large genome rearrangements in all 12 independently evolving populations. In most cases, it is unclear whether these changes were beneficial themselves or, alternatively, they hitchhiked to fixation with other beneficial mutations. In either case, many genome rearrangements accumulated over decades of evolution, providing these populations with genetic plasticity reminiscent of that observed in some pathogenic bacteria.

Acknowledgments

This work was supported by the European Union program FP7-ICT-2013-10 project EvoEvo grant 610427 to D.S., the Université Grenoble Alpes to D.S., the Centre National de la Recherche Scientifique to D.S., the U.S. National Institutes of Health grant R00-GM087550 to J.E.B., the U.S. National Science Foundation grant DEB-1019989 to R.E.L., and the U.S. National Science Foundation support DBI-0939454 for the BEACON Center for the Study of Evolution in Action. C.R. was supported by a fellowship from the French Ministry of Education and Research. D.E.D was supported by a traineeship from the Cancer Research Prevention Institute of Texas. Acknowledgments. This work was supported by grants from the European Union program FP7-ICT-2013-10 project EvoEvo grant 610427 to D.S., the Université Grenoble Alpes to D.S., the Centre National de la Recherche Scientifique to D.S., the XXXX. C.R. thanks the French Ministry of Education and Research for a doctoral fellowship.

Results

Supplemental Material

Supplemental material for this article may be found at XXXX To whom correspondence may be addressed. dominique.schneider@ujf-grenoble.fr

Abstract

Insertion Sequences (IS) are mobile genetic elements ubiquitous in bacterial genomes where they have profound impact on both structure and expression. Like point mutations, they produce harmful, neutral and beneficial effects on organismal fitness. In contrast however to mutation rates, whether and how the balance between these opposing effects affect the longterm dynamics of IS elements in bacterial genomes is unknown. Moreover, their distribution and copy number are highly variable, for unclear reasons. Therefore, it is still controversial whether IS elements are genomic parasites maintained through high transposition rates and genetic exchange or major evolutionary drivers under positive selection. Here, we investigated the long-term dynamics of IS elements and their contribution to the genomic mutagenesis during the longest-running evolution experiment, where twelve populations are propagated from an Escherichia coli ancestor for over 60,000 generations. We demonstrated that IS elements accounted for 15 to 50% of the total mutations substituted during 40,000 generations of evolution. In one population, we observed a substantial ~6-fold increase in the copy number of one IS type, IS150. By experimentally checking three evolutionary scenarios and using an evolutionary model, we show that this genomic expansion of IS150 was most likely associated with early positive effects on fitness. Later during evolution, when bacterial cells were adapted to their environment, this expansion was compensated by a down regulation of IS150 transposition activity, suggesting domestication. Therefore, the long-term dynamics of IS elements, as for mutation rates, involves a balance between robustness and evolvability.

Keywords: Insertion Sequences | experimental evolution | Escherichia coli | copy number dynamics in all 12 populations at 40,000 generations (Table S1) to the ancestor, revealing a total of 210 unique IS copies (Table S2). IS1 and IS150 were active in all 12 populations while changes related to IS3, IS4 and IS186 were detected in 8, 3 and 11 populations, respectively (Table S3). No changes were detected for IS2 and IS30. The number of IS-related changes was different in each population and unrelated to their ancestral distribution (Χ 2 = 1.09 x 10 -57 , Fig. S1). The phylogenetic tree, based on these IS fingerprints, revealed a high genetic diversity allowing to distinguish each individual evolved clone (Fig. S2). However, clones from identical populations grouped together with two noticeable exceptions: Ara-2 which contains two ecotypes (31), and Ara-6 which therefore may also contain at least two lineages.

A previous study indeed showed that co-existing lineages may be present in several populations (32).

Contribution of IS elements to total mutations during 40,000 generations. We next scrutinized the genome sequences of the same 28 evolved clones to compute the proportion of IS changes relative to all mutations. The total number of mutations was inferred from the genome sequences and the number of IS-related changes from both the IS fingerprints and genome data for comparative purposes (Fig. 1). Similar results were obtained with both datasets except for Ara-5 where 41 and 90 IS-related changes were detected for the 3 analyzed clones by genome sequencing and IS fingerprinting, respectively. We distinguished three groups of populations (Fig. 1): first, populations Ara+3, Ara+6, Ara-1, Ara-2 and Ara-4 showed a low contribution (< 5%) of IS-related changes, reflecting their mutator status and therefore a high proportion of SNPs. Second, in populations Ara+2, Ara+4, Ara+5, Ara-3, Ara-5 and Ara-6, IS-related changes accounted for ~10 to ~30% of all mutations. Third, population Ara+1 was unique in that IS-related changes accounted for almost 50% of all mutations. The proportion of IS-related changes had a median value of 0.28 in the non-Conclusions. While IS elements have been considered as genomic parasites (21) with harmful effects, evolution experiments provided evidence that IS elements can persist within bacterial genomes by generating beneficial mutations (22)(23)(24)(25)(26)(27). However, the dynamics of these opposing effects during long-term evolution is mostly unknown. In particular, how IS elements can invade bacterial genomes without extinction of their host is unclear.

Here, we investigated the dynamics of IS elements in 12 bacterial populations that evolved over tens of thousands of generations. IS elements generated a high level of genetic diversity and their contribution to total mutations was high from 15 to as much as 50% in non-mutator populations. One population experienced a large expansion of one type of IS elements, IS150. This expansion was not related to evolutionary changes in either the sequence of IS150 itself or its transposition regulation, but rather to the initial new copies that conferred most of the fitness increase during the first 2000 generations. This initial increase in IS150 copy numbers, associated with positive selection, likely drove the subsequent boost of transposition of IS150 by increasing the intracellular transposase concentration. Expansion of IS elements has been commonly reported for bacteria with host-restricted lifestyles or pathogenic bacteria, and has been suggested to be involved in the first steps of genome reduction and in the improvement of bacterial ability to fight host defences (18,28). We also recently showed that the expansion of IS150 was associated with profound restructuration of the chromosome in population Ara+1 including successive large chromosomal inversions by recombination between IS150 copies (mBio manuscript). The IS150 expansion was followed, after 40,000 generations and a 6-fold increase in copy number, by a reduced frequency of IS150 transposition, which might indicate IS150 domestication by these bacterial cells (18). This behaviour is reminiscent to the dynamics of mutation rates during long-term evolution, including a first strong increase followed by compensation to reduce genetic load (4,13). Therefore, the expansion of IS elements during Each plasmid-carrying strain was grown in 2 mL LB-Kan at 28 °C for 48 h, diluted and plated onto two sets of agar plates, LB-Kan-Cam and LB-Cam, which were then incubated at 28 °C and 42 °C, respectively. We scored the total number of cells carrying pFDX2339 (N T ) as the number of colonies growing on the first set of plates, and the total number of cells that integrated into the chromosome either the entire plasmid by recombination (N R ) or the modified IS150 copy by transposition (N IS ) as the number of colonies growing on the second set of plates. To distinguish between the two last types of events, colonies that grew at 42 °C on LB-Cam plates were streaked onto both LB-Kan-Cam and LB-Cam plates and incubated overnight at 42 °C. Growth onto both plates gave (N R + N IS ) while growth onto only LB-Cam gave N IS . Finally, the proportion of cells that experienced IS150-mediated recombination and transposition was calculated as the ratios N R /N T and N IS /N T , respectively.

Contribution of IS elements relative to total genomic changes. The proportion of ISmediated changes was calculated as the ratio (number of IS-mediated mutational events) / (total number of mutations). The mean of the values obtained for the evolved clones from the same population was then calculated. The number of IS-mediated changes was derived from both the IS fingerprints and the genome sequences. The total number of mutations sums the number of SNPs identified in its genome sequence and the number of IS-mediated changes.

We gave equal weight to losses and gains of IS copies and we did not adjust for simultaneous loss and gain of hybridizing bands from single mutational events.

Construction of isogenic mutant strains and competition experiments.

We constructed an in-frame deletion of each of the nadR and hokB loci in the ancestral chromosome by allelic exchange. All gene replacement experiments were performed by using suicide plasmid pKO3 as described (34). To construct in vitro the 300-bp hokB in-frame deletion, we PCR-amplified the hokB upstream and downstream regions using primer pairs G125 ( 5 Table S6. Data used to calculate IS150 recombination and transposition frequencies.

Ancestor ( 1 S1), and is given for each IS element according to the indicated colour code. The 12 populations are shown by their names (Ara+1 to Ara+6 and Ara-1 to Ara-6) with stars indicating those that evolved increased mutation rates compared to their ancestor. Two populations (Ara+1 and Ara-3) revealed a strong increase (≥ 30) in IS-related changes, seven (Ara+2, Ara+5, Ara-1, Ara-2, Ara-4, Ara-5 and Ara-6) a moderate increase (15 to 30), and three (Ara+3, Ara+4 and Ara+6) a lower increase (< 15).

General Discussion

Diversity of events and potential effects

One common result was that there were large differences in the extent that each population was affected by rearrangements or IS elements. For rearrangements all the twelve populations but one sustained more than five rearrangements and four of these populations more than ten.

The average number of rearrangement is 7.5. The extent of these rearrangements is variable as some population (Ara+1 and Ara+2) sustained reorganization of more than the half of the chromosome whereas some other as Ara+4, Ara+5, Ara+6 show small changes.

There is no clear link between the number and the extent of chromosome rearrangement.

Population Ara+5 sustains the lowest number and extent of rearrangement, on the other end the population Ara-3 with 20 rearrangement is the most heavily affected but with a limited impact on the overall organization of the chromosome. Similarly IS elements have varying impact on the populations. Two populations, Ara+1 and Ara-3, had high levels of IS participation in the rearrangement events while other populations varied between 30 and 12 changes. This variety in IS copy numbers and in the IS distribution can be explained by the random method of IS dissemination throughout the genome. IS insertions account for ~50% of all mutations in the population Ara+1 while in other non-mutator populations it varies between 35% and 20%. Large differences in contribution of IS changes have been previously observed in experimental evolution, with batch cultures (Rhiele, Bennett et al. 2001 ) and chemostat cultures [START_REF] Maharjan | A case of adaptation through a mutation in a tandem duplication during experimental evolution in Escherichia coli[END_REF] or in natural isolates [START_REF] Kresse | Impact of large chromosomal inversions on the adaptation and evolution of Pseudomonas aeruginosa chronically colonizing cystic fibrosis lungs[END_REF], Rhomer, Fong et al. 2007). The IS insertions detected are generally those that have been fixed in a population and may represent insertions that have either a beneficial or neutral effect on the cell. There was no correlation between the number of IS element changes and the number of rearrangements. Even though it can be supposed that the number of IS elements would increase the number of available targets for recombination. Major phenotype changes occurred in several populations leading to the cit+ phenotype in Ara-3 (Blount, The main effect of chromosomal imbalance caused by inversions is a change in chromosomal replication times as seen in other evolution experiments. In a short evolution experiment performed in E. coli O157:H7, several chromosome inversions occurred in the vicinity of the Ter region [START_REF] Iguchi | Spontaneous recombination between homologous prophage regions causes large-scale inversions within the Escherichia coli O157:H7 chromosome[END_REF]) most of these inversion affect the genome balance of 30 to 83 kb. However, one inversion induced a significant unbalance of the replichore, close to 7.5% of the genome [START_REF] Iguchi | Spontaneous recombination between homologous prophage regions causes large-scale inversions within the Escherichia coli O157:H7 chromosome[END_REF]). However cells dividing in the a minimum media depleted in glucose conditions used during the LTEE, , have a generation time of over 3.12hours and allow an off-balance of 10% of the total genome to be still viable.

Conclusions and Perspectives

These data shows that rearrangements and insertion sequences have an important role in the adaptation of an organism to its environment due to the large range of genomic flexibility accorded. Both event types can create a varied mutation spectre including large ranging effects over entire operons. While the actual fitness gain of each rearrangement is unknown it can be speculated that, for at least, the parallel events give an advantage to the cell. The participation of IS elements is important as it shows that they have a more dynamic role in the cell than just gene interactions. These results also show that the populations are not clonal but rather heterogeneous.

This work focused on the evolution of rearrangements and insertion sequences (IS) in the longest ongoing evolution experiment (LTEE). Both rearrangements and IS play vital roles in the evolution of the populations. To continue this work it is essential to elucidate the exact benefits that are brought by the mutations. Some mutations have already been studied in previous works like the deletion of the ribose operon. Other deletions such as the rearrangement between manB-cpsG are candidates for investigation as it is deleted in ten out 180 of the twelve populations. A duplication worth investigating is the duplication containing rpoS found in two of the twelve populations by optical mapping. Since this study has mainly focused on IS movements in the population Ara+1, the dynamics of IS elements is worth investigating in other populations. On a longer term it is possible to deconstruct the rearrangements and the mutations in some evolved clones, focusing on Ara+1 at 2000 generations, and to replay the evolution experiment to see if the evolutionary trajectory is the same.