Xavier Veaute

Bénédicte Michel

Michael Dubow

Aurélie Bobillot

Mélanie Guillemet

Renata Matos

Laureen Crouzet

Abdel Boukadiri

Aline Viel

Annie Mathieu

Emmanelle Bourneuf

Deborah Jardet

Elisabetta Giuffra

Evelyne Lhoste

Olivier Telle

Setha Ketavong

Pierre-Yves Holtzmann

Elodie Poumerol

Guillaume Piton

Ce que m'a appris ce doctorat sur la recherche peut se résumer par le célèbre adage d'Edward A. Murphy Jr., plus connu sous le nom de loi de Murphy, « Anything that can go wrong, will go wrong » (« Tout ce qui peut mal tourner va mal tourner »). Effectivement, la plupart des expériences menées en laboratoire se soldent par des échecs. Et des échecs j'en ai essuyé de nombreux avant d'arriver au rendu final que vous allez lire. Fort heureusement, on apprend de ses échecs, on peut alors se relever et recommencer grâces aux conseils, aux remontrances ou au réconfort des gens qui nous entourent, à la fois dans notre vie professionnelle et privée, afin de réussir à mener un projet jusqu'à son terme. C'est pourquoi je souhaite remercier les personnes qui m'ont permis de mener à bien ce projet de thèse, et de rentrer dans cet étrange, mais merveilleux, monde qu'est la recherche. Tout d'abord, je souhaite remercier les membres de mon jury de thèse, chargés de m'évaluer pour l'obtention du grade de docteur. Merci à Mart Krupovic et Stéphanie Marsin qui ont accepté de relire et juger mon manuscrit en tant que rapporteurs, et surtout merci pour leurs commentaires plus qu'encourageants. Merci à François Enault et à Mauro Modesti d'avoir accepté d'être examinateurs. Enfin merci à Cécile Fairhead, la présidente du jury.

Je souhaite ensuite remercier les membres de mon comité de suivi de thèse, qui m'ont aidé chaque année avec leurs remarques pertinentes et pour les discussions passionnantes qui m'ont permis d'avancer. Merci donc à

Material and Methods

Results

Temperate phage encoding Sak4 often encode an atypical SSB nearby

The SSB HK620 is essential for Sak4 HK620 SSA activity in vivo Both Sak4 HK620 and Redβ λ anneal up to 12% diverged sequences in vivo

Table of figure contents

Chapter I: Figure 6: GST-Sak4 PA73 does not promote strand exchange. This introduction will first give an overview of phage genome structures, compared to bacterial genomes. In particular the concept of phage genome mosaïcism will be explained. The hypothesis guiding my doctoral research has been that the great facility of genetic exchanges among phage genomes may be due in part to the particular propriety of phage proteins that catalyse homologous recombination.

The key proteins for homologous recombination are the recombinases. A second part of this introduction will therefore summarize what was known on bacterial and phage recombinases at the onset of my work. The particularity of the bacterial machinery is the extreme fidelity of the Homologous recombination system. Indeed, this system permits to maintain the genome integrity when some stresses induce DNA damages. In phages, the homologous recombination machinery is not as faithful as that of Bacteria. Indeed, a 2008 paper has shown that exchange of genetic material catalyzed by λ phage recombinase occurs even between two 22% diverged sequences. However, whether this unfaithful property was restricted to the λ phage or common among phage recombinase was no known. The position of the root was determined by comparing sequences of pairs of paralogous genes that diverged from each other before the three primary lineages emerged from their common ancestral condition. The numbers on the branch tips correspond to the following groups of organisms. Bacteria: 1, the Thermotogales; 2, the flavobacteria and relatives; 3, the cyanobacteria; 4, the purple bacteria; 5, the Gram-positive bacteria; and 6, the green nonsulfur bacteria. Archae: the kingdom Crenarchaeota: 7, the genus Pyrodictium; and 8, the genus Thermoproteus; and the kingdom Euryarchaeota: 9, the Thermococcales; 10, the Methanococcales; 11, the Methanobacteriales; 12, the Methanomicrobiales; and 13, the extreme halophiles. Eucarya: 14, the animals; 15, the ciliates; 16, the green plants; 17, the fungi; 18, the flagellates; and 19, the microsporidia. [START_REF] Woese | Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya[END_REF] Bacterial genomes have begun to be completely sequenced and compared at the eve of the 21 st century. However, the task of classifying them, as has been done for the Eukaryotes, turned out to be more complicated than expected. Classification has been successively based on their phenotypes, their capacity to cross hybridize their chromosomal DNA, their 16S-rDNA sequences, their gene content proximity, or their average nucleotide identity (ANI). However, bacterial species boundaries are still unclear and discussed [START_REF] Konstantinidis | The bacterial species definition in the genomic era[END_REF]. The reason for this perplexity is the incredible plasticity of bacterial genomes due to horizontal gene transfers. Indeed at the species level, we can distinguish genes of the core-genome, comprising genes shared by all (or almost all) strains within a species, and those from the pan-genome, comprising the constant and the variable gene pool, made of genes found in only some of the strains of a given species. There seems to be no correlation between the gene function and its belonging or not to the core genome [START_REF] Huang | Comparative bacterial genomics: defining the minimal core genome[END_REF]. It is nevertheless recognized that prophage genes constitute a large fraction of a species pan-genome (15% in Salmonella enterica, 30% in Escherichia coli, (Bobay, Rocha, Touchon 2013)). Whole genomes can be aligned at the species level thanks to their core-genome, and partitioned into a conserved backbone, interrupted by a collection of variable segments (Figure 2, [START_REF] Chiapello | Systematic determination of the mosaic structure of bacterial genomes: species backbone versus strain-specific loops[END_REF]). Variable segments have various lengths: the large ones correspond to prophages and other genomic islands, the small ones to intra-genic microdiversity [START_REF] Touzain | Small variable segments constitute a major type of diversity of bacterial genomes at the species level[END_REF].Extraction of the species backbone is useful for the detection of small DNA motifs used by bacteria for the repair of the chromosome (Chi sites), or its segregation (KOPS sites, MatS sites, [START_REF] Touzain | DNA motifs that sculpt the bacterial chromosome[END_REF]. It seems therefore that the last 15 years of intense sequencing efforts have permitted to reach a good overview of bacterial genomes, where for most species a common coregenome is observable, whose genes can be used for deriving robust phylogenies [START_REF] Touchon | Organised genome dynamics in the Escherichia coli species results in highly diverse adaptive paths[END_REF]). Strain-specific variable segments that correspond mainly to the gene flux inside a species interrupt this backbone. These segments are exchanged among strains by horizontal transfer, or loosed by deletion. clockwise, all EGD-e genes are arranged in chromosomal order. Continuing clockwise, all genes not present in EGD-e are grouped by strain (as indicated by segment labels). Genes in the F2365 segment are present in F2365, but absent from EGD-e, and genes in the Clip81459 segment are present in Clip81459, but absent from F2365 and EGD-e, and so on. In this way, each gene is represented only once in the diagram. Gene order in all segments except EGD-e is monotonically increasing, but discontinuous, since shared genes may be represented in other segments. Internal circles indicate gene presence (solid color) or absence (unfilled) of each gene in each of the 13 strains examined.

Circles from outer to inner are in the same order as strains on the outer circle, starting with EGD-e, followed by F2365, etc. L. monocytogenes strains are in blue; L. marthii is in green; L. innocua strains are in gold; L. welshimeri is in orange; L. seeligeri strains are in red; L. ivanovii subsp. londoniensis is in purple. The location, in the EGD-e genome, of the prfA virulence cluster, conjugative transposon tn916 and prophage A118 are specifically indicated. (den Bakker et al. 2010)

II) Bacteriophage genomes

Bacteriophages are viruses infecting Bacteria. They are generally specific to a species, but some infect bacteria belonging to the same genus, or others are restricted to some strains into the same species.

1) History and definition

The first observation of bacteriophages was reported by Frederick Twort in 1915 (Twort 1915). He described a "transparent material" that grew on a lawn of a Micrococcus strain. He called it ultramicroscopic (i.e. beyond detection under the microscope) viruses. He observed that these viruses could not grow alone, and could not grow on other bacteria, animals, neither human.

Felix d'Herelle isolated an anti-Shiga microbe (named Shigella today) in 1917, noted that it lyses liquid Shiga culture, and made on Petri dishes "a certain number of circles of around 1 mm in diameter on which the culture is non-existent". From this, he concluded that the anti-Shiga is not a chemical compound but a life form. He could not isolate this invisible microbe alone in a culture medium, thus he concluded this anti-Shiga was an "obligate bacteriophage". "Bacteriophage" (phage in short) was then coined as the name used to define viruses that grow on bacteria (D 'Herelle 1917).

There are two categories of phages: virulent and temperates (Figure 3). Both infect Bacteria and inject their genome from the virion into the bacterium. Both kinds can undergo a lytic cycle, whereby genomes are rapidly replicated and encapsidated. The bacterium is lysed to release the virions formed, or phages are secreted from the bacterium in the case of filamentous phages such as M13 [START_REF] Beaudoin | Antiserum Inactivation of Electrophoretically Purified M13 Diploid Virions Model for the F-Specific Filamentou[END_REF]. In addition, temperate phages can insert their genome into the bacterial chromosome instead of replicating themselves. Almost all genes are repressed during this cycle. The phage is then named a prophage or lysogenous phage. The dormant prophage is replicated passively during bacterial multiplication, and can excise itself, if induced by an endogeneous or exogenous stress, to start again a lytic phase.

Phages are very difficult to classify. The first manner to classify them is based on the type of genetic material present in the virion. It can be DNA or RNA, single strand or double strand. However, most bacteriophages known today are dsDNA, and their virion has a tail (Caudoviridae). A subclassification is then made among Caudoviridae, based on the tail type: short for Podoviridae, long and non-contractile for Siphoviridae, and long and contractile for Myoviridae (ICTV classification, last update in 2013). However, this sub-classification is not monophyletic for some groups, such as Mulike phages, that are closely related but can be either Sipho-or Myoviridae. Genomics analyses are Chapter I: Introduction, Bacteriophage genomes, History and definition 20 now on the front stage for virus classification. Rohwer and Edwards were the first in 2002 to try to construct a proteomic tree from 105 completely sequenced phage genomes [START_REF] Rohwer | The Phage Proteomic Tree: a Genome-Based Taxonomy for Phage[END_REF]. They used a distance based on the inverse of the number of genes shared by two phages.

However, their branch history did not fit with the capsid classification. Lima-Mendez et al. had a similar approach on 306 completely sequenced phage genomes, except that they eliminated the hierarchical tree-like approach [START_REF] Lima-Mendez | Reticulate representation of evolutionary and functional relationships between phage genomes[END_REF]. They looked for pairwise similarities and drew a network to cluster phages. They obtained separate graphs for ssDNA and RNA phages, and most Caudoviridae were connected into a single network, with temperate phages occupying the center of the network, and virulent phages (such as T4, T7) forming more distant 'segregating' nodes.

However, the difficulty with network representations is their fluidity: the network is expected to change when more genomes are added, so it may not be usefull for classification matters. Even if no universal gene such as the 16S rDNA gene is present among phages, single gene approaches have been tried on sub-categories of phages, such as Caudoviridae, that share a related major capsid protein, but also portal and terminase genes that are well conserved [START_REF] Smith | Evolutionary relationships among actinophages and a putative adaptation for growth in Streptomyces spp[END_REF]. Very recently, a classification based on the retrieval of a collection of 3 to 9 proteins involved in the connection between the virion head and tail has been proposed, that permits to classify >90% of the completely sequenced Caudoviridae (Lopes et al, submited). This classification identifies several Virulent phages enter in the lytic cycle: the host genome is sometimes degraded (as represented here), and the phage multiplies itself. Capsid proteins are expressed and assembled. The phage genetic material is then encapsidated and the lysis of the cell releases virions. Temperate phages have another option after genome injection. They can enter into the lysogenic cycle instead of the lytic cycle in certain conditions. The phage genome is integrated into the bacterial genome and replicated at the same time as the bacterial chromosome. The phage is then said to be in a prophage state. After a stress, the prophage is induced and the phage enters in the lytic cycle.

groups that are closely related phages, despite having Myo or Siphoviridae virions, as observed for Mu-like phages. It may be that comparative genomics will finally help reaching a satisfying method of classification for phages.

2) Genome organization A) Virulent phages

Virulent DNA phages possess either very large genomes, in the 80-200 kb range, or very small ones (below 20 kb). Those of the T4 family belong to the large genome category; they have been largely sequenced, and compared to one another. They have a core genome, and the shared genes can be used to build evolutionary trees [START_REF] Sarker | Oral T4-like phage cocktail application to healthy adult volunteers from Bangladesh[END_REF]. The rest of the genome of T4-like phages is hyper-plastic and encodes for genes probably implied in the adaptation to the host (Figure 4, regions that are not grey shaded) [START_REF] Comeau | Modular architecture of the T4 phage superfamily: a conserved core genome and a plastic periphery[END_REF]). In fact, many of these genes have unknown functions. Somehow, the structure of T4-like genomes is reminiscent of that of bacteria, with a common backbone and phage-specific variable segments. Genome maps of the phages are drawn to scale and, in each, the outer rings show the scales (in kb), the middle rings (yellow arrows) the T4 homologues, and the inner rings (red arrows) the ORFs with no homology to T4. T4 genes that have been rearranged (radiating blue bars) are indicated in their new locations. Various T4 genes are given as landmarks on the outside of the rings and the hyperplastic regions are indicated. The virion modules, tail fiber genes, replication modules and tRNA cluster are indicated by gray boxes. [START_REF] Comeau | Modular architecture of the T4 phage superfamily: a conserved core genome and a plastic periphery[END_REF])

A recent study showed that T4-like Cyanobacteria phage genomes are prone to high levels of horizontal transfer, probably mediated by homologous recombination (Ignacio-Espinoza, Sullivan 2012). Indeed, the T4 group of phages possesses genes encoding for a homologous recombination system. This system is similar but independent from that of the host (see below for further explanations).

B) Temperate phages

Temperate DNA phages fall into two genome categories: small sized genomes (4-10 kb range), usually encapsidated as ssDNA [START_REF] Roux | Evolution and diversity of the Microviridae viral family through a collection of 81 new complete genomes assembled from virome reads[END_REF], and medium sized genomes (20-80 kb range), usually forming dsDNA virions. This last category has a genomic structure completely different from that of T4-like phage genomes. It is impossible to delineate a shared backbone even among related genomes: rather, each genome seems to be composed by an assemblage of parts coming from recent horizontal transfers. The phage genomes are said to be "mosaic" ( (Brussow, Canchaya, Hardt 2004) for review, Figure 5) The open reading frames are color coded according to their predicted function (Green, predicted DNA packaging and head morphogenesis genes; brown, predicted head-to-tail joining genes; dark blue, tail morphogenesis genes; light blue, tail fiber genes; mauve, lysis genes; black, predicted lysogeny conversion genes; red, lysogeny genes; orange, DNA replication genes; yellow, transcription regulation genes ). Selected genes or genomic features are denoted. Genes encoding proteins that showed significant amino acid sequence are linked by red shading, and the percentage of amino acid identity is indicated. The degree of amino acid identity (>90, >80, >70, or <70%) is reflected in the color intensity of the red shading. Inspired from [START_REF] Proux | The Dilemma of Phage Taxonomy Illustrated by Comparative Genomics of Sfi21-Like Siphoviridae in Lactic Acid Bacteria[END_REF].

The Hatfull team, helped with students, has been isolating numerous mycobacteriophages and sequencing their genomes. In 2011, 62 genomes were sequenced and compared. Each phage can be classified into one of 11 super groups depending on its genome structure and sequence homologies.

Interestingly, although groups can clearly be delineated, genetic exchanges are observed between groups ( [START_REF] Pope | Expanding the diversity of mycobacteriophages: insights into genome architecture and evolution[END_REF]. Recently, an upgrade with 471 phages led to the addition of 8 new super groups [START_REF] Hatfull | Mycobacteriophages: windows into tuberculosis[END_REF]).

These authors think that genetic exchanges among phages are mainly due to illegitimate recombination: two phages in the same bacterium break and ligate their genomes to form a new viable phage. Other argued on the possibility that phage recombinases (see below) may play a role in these exchanges, because of their unique propriety to recombine diverged sequences, as described below (Martinsohn, Radman, Petit 2008).

Mosaicism among temperate dsDNA phages of Staphylococcus is remarkably extant [START_REF] Kwan | The complete genomes and proteomes of 27 Staphylococcus aureus bacteriophages[END_REF]. A recent study tried to recreate the evolutionary history of 31 Staphylococcus aureus phages with the hypothesis of homologous recombination [START_REF] Swenson | Reconstructing the modular recombination history of Staphylococcus aureus phages[END_REF]). To do so, they truncated these phage genomes into segments. Each sequence of a segment is classified as an allele, called here variant, based on its identity (at least 94% of identity to belong to a group of variants, and at most 76% identity with any other variant). The basic postulate is that between two identical variants of the same segment, homologous recombination can occur and form a new mosaic phage from the two ancestral phages. They were able to observe these events and to recreate three ancestral phage genomes from the isolated phages, which exchanged genetic material with other phages.

3) Ways to exchanges genomes fragments

Different molecular processes will be described here, that can explain the mosaic formation among temperate phages. They will be presented starting from the mechanisms requiring little or no homology between the exchanged fragments (the broad "illegitimate recombination" category), up to those involving more extant homology stretches. Interestingly, in the phage world, the frontier between these two classical categories seems to be somewhat blurred.

A) Polymerase slippage

DNA polymerase can switch from a matrix to another if some homology (superior to 14nt) is present between the two ends of the recombination event. A model proposes that the polymerase stops due to the presence of a block, such as a hairpin structure. The replicated strand unwinds from its matrix, and if complementarity to the tip of the replicated strand is present downstream of the pausing site, annealing occurs. DNA polymerase binds again to the replicated strand extremity and resumes polymerisation ( [START_REF] Viguera | Replication slippage involves DNA polymerase pausing and dissociation[END_REF], Figure 6A). Poteete has proposed that phage recombinases play a role in conjunction with polymerase slippage: by catalyzing the annealing of a phage 3'-ssDNA extremity with the lagging strand of the replication fork. The leading strand polymerase slips from the initial matrix to the phage strand, thanks to its close vicinity ( (Poteete 2008), Figure 6B).

B) NHEJ (Non-Homologous End Joining) NHEJ (Figure 7) is a mechanism of double strand break DNA repair well known in Eukaryota. A protein, called Ku, binds to the two extremities of a double-strand break. A machinery is then recruited to get the two broken parts closer. Finally, a ligase ligates the two DNA (see [START_REF] Ochi | The spatial organization of non-homologous end joining: from bridging to end joining[END_REF] for review). No homology between the two broken ends is needed to perform NHEJ, and this process is often associated with mutagenesis or large rearrangements. Ku orthologs were found in Bacteria [START_REF] Bowater | Making ends meet: repairing breaks in bacterial DNA by nonhomologous end-joining[END_REF]. These genes are most widely distributed in Proteobacteria (α, β, γ, and δ families), Firmicutes, and Actinobacteria. There is no evidence whether these genes are acquired by horizontal transfer or if they were present in the common ancestor and disappeared from some bacterial branches. This bacterial Ku forms homodimers that bind to dsDNA extremities. The complete NHEJ activity was not proved in bacteria and deletion of the Ku-like protein is not deleterious for UV resistance (a classical test for repair of DNA break systems). In Bacillus subtilis, in addition to Ku, some essential homologs to Eukaryotic NHEJ machinery were identified: a ligase and a polymerase (de Vega 2013).

Ku orthologs were also found in phages (d'Adda di Fagagna et al. 2003). They share more identity with bacterial Ku than with Eukaryotic Ku. In phages, this protein is Gam and was first identified in the Mu phage. For the sake of clarity, as a different Gam protein is present in λ, the Mu type of Gam will be designated as Mu-Gam hereafter. This protein interacts with DNA as Eukaryotic Ku. There is no evidence of NHEJ function in phages carrying Mu-Gam, but it is still a possibility than could explain the mosaisism.

C) MMEJ (Microhomology-Mediated End Joining) MHEJ (Figure 8) is a another way to repair double strand breaks involving an annealing step between the two ends, between small repeats of less than 10 pb. E. coli can promote this type of events at a low frequency of 10 -5 [START_REF] Chayot | An end-joining repair mechanism in Escherichia coli[END_REF]). The authors transformed E. coli with a digested plasmid, mimicking a double strand break. They showed that the RecBCD exonuclease is essential for the reaction. RecBCD resects DNA at the DSB and exposes microhomologies that anneal each other, independently of the host recombinase RecA. Then the annealing product is ligated. An alternative , where it may serve as an end-bridging and alignment factor. Following binding to the broken DNA ends, additional processing enzymes are recruited by Ku to the break site (step 2). Ku may translocate away from the ends, allowing access by other factors to the break termini.

When DNA ends are non-complementary and/or are damaged, the DNA end-processing, gap-filling, and nucleolytic activities generate DNA termini, capable of being ligated, prior to ligation (step 3). Subsequently, the broken ends are joined by an NHEJ-specific DNA ligase (step 4) and the NHEJ complex dissociates. [START_REF] Bowater | Making ends meet: repairing breaks in bacterial DNA by nonhomologous end-joining[END_REF] MHEJ pathway has been proposed, which involves RecQ, a DNA helicase. This pathway can only be seen in an E. coli strain with a recB mutation, that leads to the loss of the RecBCD exonuclease activity but maintains the helicase activity [START_REF] Ivankovic | DNA end resection controls the balance between homologous and illegitimate recombination in Escherichia coli[END_REF]. RecQ would participate to the annealing of microhomologies. Due to it slow efficiency, MHEJ is a minor pathway in E. coli, that may take place when RecBCD exonuclease activity is inactivated and no sister chromatid, or no other homologous sequence, is available to perform a RecA-dependent repair.

In an assay based on the selection of lambda prophage imprecise excision events (a process occurring in the absence of the prophage integrase gene), RecOR was also found to facilitate MHEJ, by annealing microhomologies [START_REF] Shiraishi | Roles of RecJ, RecO, and RecR in RecET-Mediated Illegitimate Recombination in Escherichia coli[END_REF]. In the same study, the authors also reported that the RecET pathway stimulates MHEJ by a factor of seven. These proteins are encoded by rac, a defective prophage in E. coli. The recET genes are normally silent, but become activated by a sbcA mutation, that brings a promoter upstream of recET. RecT is a recombinase, that performs single strand annealing (SSA) of complementary DNA sequences (see below), and RecE is a 5'-3' dsDNA exonuclease. They also saw that RecJ, a 3' to 5' ssDNA exonuclease, is essential in this reaction. Later, the same team found that the RecET pathway was inhibited by an uvrD mutation. They proposed that the UvrD helicase prevents the RecA loading and so enhances the illegitimate recombination by the RecET pathway [START_REF] Shiraishi | The role of UvrD in RecET-mediated illegitimate recombination in Escherichia coli[END_REF]. These studies suggest that phage recombinases such as

RecT may promote illegitimate recombination by MHEJ.

D) HR (Homologous recombination)

Homologous recombination should not be confounded with site-specific recombination. In this last process, a specific DNA site is recognized and recombined by a resolvase or an integrase. Most temperate phages encode an integrase, which permits integration of phage in the host genome at an attB sequence (see the review in Annex). Homologous recombination is not sequence-dependent, but relies on the homology between the DNA partners that recombine. Adapted from [START_REF] Chayot | An end-joining repair mechanism in Escherichia coli[END_REF].

In 2010, phages genomes were analyzed to identify gene encoding protein catalyzing the homologous recombination events, named recombinases. Among 365 dsDNA phage genomes of a size superior to 20Kbp, 60% contained a recombinase (Lopes et al. 2010). By detecting distant homology between amino acid sequences, all recombinases were classified into three super families, Rad52, Rad51 and Gp2.5-like proteins (these super-families are described in details later in this chapter). The crystallographic structures for members of Rad51-like and Gp2.5-like recombinases have been solved (Hollis et al. 2001;[START_REF] Gajewski | Crystal structure of the phage T4 recombinase UvsX and its functional interaction with the T4 SF2 helicase UvsW[END_REF]. None of any phage Rad52-like recombinase has been crystallized to sufficient resolution up to now to permit 3D reconstitutions.

The Redβ 3D model below is taken from Lopes et al. (Lopes et al. 2010). It was model on the human Rad52 Nt domain, which has been cristalized [START_REF] Kagawa | Crystal structure of the homologous-pairing domain from the human Rad52 recombinase in the undecameric form[END_REF])(Figure 9). Several phage recombinases, belonging to the Rad52 and Gp2.5 families, are known to promote the single strand annealing (SSA) reaction ( (Kmiec, Holloman 1981;Rezende et al. 2003;Datta et al. 2008;Ploquin et al. 2008) see paragraph III of this chapter for further explaination).

Some phages infecting Enterobacteria also encode for proteins that inhibit the main host exonuclease, RecBCD. This is a way to protect the phage rolling-circle intermediates of replication from degradation (Figure 10). The first one to be reported was Gam, from phage λ, that infects E.

coli. The Gam protein was crystallized and looks like a globule with two mobile α-helixes that have the same diameter as ssDNA. The hypothesis is that at least one of the two helixes takes the place of ssDNA in RecBCD complex, inhibiting its DNA degradation activity (Figure 10, (Court et al. 2007)).

Phage Phi80 encodes GamL, which performs the same function as Gam, while sharing no homology with the Gam protein (Rotman et al. 2012). Phage P22 encodes a third type of RecBCD modulator, Abc2, (see part III of this Manuscript) that only partially inhibits the ds exonuclease, and is proposed to convert it into a ssDNA 5'-3' exonuclease (Murphy 2000). Finally, the temperate phage Mu encodes a fourth form of RecBCD 'protector', Mu-Gam, (d'Adda di Fagagna et al. 2003), which binds to the extremities of the phage genome, and thereby prevents their degradation by RecBCD. This protein has already been mentioned in paragraph 2B, as it shares homology with Ku proteins.

Whether phages infecting Gram positive bacteria also encode Gam like functions is unknown at present. Given the panel of diverse proteins performing similar functions, the possibility exists that this activity is also present among temperate phages infecting Gram+ bacteria, but remains unveiled until now. We can ask why phages encode their own recombinase, since almost all bacterial hosts already encode one. This question has been addressed in 2013 for 275 prophages extracted from genomes of Enterobacteria (Bobay, Rocha, Touchon 2013). They compared the occurrence of (1) phage recombinase gene, (2) a phage gene coding for Gam-or Abc2-like inhibitors of RecBCD, and (3) the presence on the phage genome of Chi sites (GCTGGTGG, a site recognized by RecBCD, which converts the potent double strand exonuclease into an ssDNA exonuclease and RecA loader [START_REF] Smith | How RecBCD enzyme and Chi promote DNA break repair and recombination: a molecular biologist's view[END_REF].

Interestingly, and contrary to what was generally suposed, they found that a majority of the prophages had Chi sites. They also reported evidence for a clear counter selection for Chi-sites on prophages encoding a Gam or Abc2-like inhibitor of RecBCD. They proposed that phages had to choose between the host recombination system (i.e. accumulate Chi sites) and their own system (i.e.

encode both a RecBCD inhibitor and a recombinase). Nevertheless, their analysis did not permit to conclude that. First, some of the prophages used in this analysis are defective, and no exact assessment of the proportion of active phages among their set was performed. Furthermore, some RecBCD inhibitors might have been missed, as they looked for only two of them: Gam and Abc2.

Finally, their analysis uncovered a significant category of prophages (20% of the set) encoding both a recombinase and Chi sites, suggesting that phages are not in an "either-or" choice between the two recombination systems. Indeed, we have shown (see Chapter II) that phage λ recombines with prophages using independently either the host RecA and its own Redβ system (redundancy), with equal efficiency when the homology region is long enough (2 kb). Interestingly, in λ, no Chi sites are present. This suggests that the RecA-dependent recombination occurs independently of RecBCD, probably by the RecFOR pathway. An alternative explanation to the observations of Bobay et al. is that defective phages, in their decay process, acquired Chi sites and lost some part of their genome, potentially containing a recombinase and a RecBCD inhibitor.

Their analysis shows notwithstanding that phages carrying a recombinase have genomes bearing traces of more recent exchanges than recombinase-less phages, suggesting that phage recombinases facilitate DNA exchanges. Our own analysis of phage mosaics has also uncovered traces of homologous recombination at the vicinity of 40% of all recent exchanges (see Chapter II).

Other clues on the particularities of phage-encoded recombinases can be suggested by interaction maps. The interaction map of the λ proteins has been explored [START_REF] Rajagopala | The protein interaction map of bacteriophage lambda[END_REF] and interactions between its recombinase Redβ and P, N, Int and A were found. P is the replicative helicase loader, and such an interaction may help to load a replication fork outside of the origin, in a process linked to recombination. However, a compared analysis of replication yield/products of λ in the presence and absence of Redβ indicated no loss of products in the mutant, and only marginal changes in the proportion of circular and sigma-shaped structures [START_REF] Better | Studies on the replication of Escherichia coli phage lambda DNA. I. The kinetics of DNA replication and requirements for the generation of rolling circles[END_REF]. N is an Chapter I: Introduction, Homologous recombination, History and definition 30 anti-termination protein, which permits transcription past early terminator region. How Redβ may facilitate N action is not immediately evident, and the interaction may be an artifact. Int is the sitespecific integrase, it is curious Redβ interacts with it. It suggests that site-specific recombination and homologous recombination may somehow be linked. A is the DNA large sub-unit of the Terminase of λ. It fills capsids until recognition of a cos site on λ genome, which it cuts. However, the packaging cannot continue if the cos site is not complete, for instance if the DNA molecule is a monomer of the genome. It is possible that Redβ, through its interaction with the terminase, is brought to the capsid in such cases, and reanneals two cos sites to continue the packaging.

III) Homologous recombination

My research during this PhD has focused on homologous recombination, especially that mediated by phage recombinases. I will therefore summarize here what is known on the bacterial host homologous recombination system before those of phages.

1) History and definition

The comprehension of homologous recombination began long before the understanding of DNA structure and gene concept. Mendel proposed a model in 1865 for the segregation of characters from a generation to another, where each character segregates independently [START_REF] Mendel | Versuche über Plflanzen-hybriden. Verhandlungen des naturforschenden Ver-eines in Brünn Bs[END_REF]. Later,

Morgan understood that the characters encoded by genes are carried by chromosomes and so can be genetically linked [START_REF] Morgan | Chromosomes and Associative Inheritance[END_REF]. Muller showed a synaptic attraction between homologous chromosomes and saw chromosome rearrangements after X-ray induced breaks [START_REF] Muller | The Frequency of Translocations Produced by X-Rays in Drosophila[END_REF].

Since the discovery of DNA structure [START_REF] Watson | The structure of DNA[END_REF], some molecular models for recombination were published (all models are presented in Figure 11). The following summary is inspired by the historical review of Haber [START_REF] Haber | RecA family proteins in archaea: RadA and its cousins[END_REF]). The first model to be published sought to explain recombination between a broken and an intact λ phage molecule (Figure 11A, [START_REF] Meselson | Chromosome brekage accompanying genetic recombination in bacteriophage[END_REF]). They thought that each strand of the broken DNA anneals to an intact complementary DNA, which serves as a template for DNA polymerization. Holliday conceived another model in 1964

(Figure 11B). He proposed that recombination begins by a coordinated pair of single-strand nicks on compatible strand pairs of homologous chromosomes. The nicked strands unwind and move to the other strand to form a single strand exchange. This leads to a four-stranded structure called now Holliday junction. This junction is symmetrical and can be then resolved by two cleavages that yield different products: (1) one cleavage gives an exchange between the two chromosomes, the so-called crossing-over. (2) The symmetrical cleavage leads to a small exchange of the single strands annealed previously, called non-cross over, or patch repair [START_REF] Holliday | A mechanism for gene conversion in fungi[END_REF].

In 1966, Thomas proposed another mechanism, whereby 4 nicks are formed at different places, DNA strands are unwound so that ssDNA is produced. If this ssDNA finds a complementary ssDNA, an heteroduplex DNA can be formed (Figure 11C, [START_REF] Thomas | Recombination of DNA molecules[END_REF]). This mechanism is now known as single strand annealing (SSA). Later on, it was proposed that only one of the two DNA molecules receive one nick (Figure 11D, [START_REF] Meselson | A general model for genetic recombination[END_REF]). The 3' end could serve as a primer for DNA polymerization and displace the complementary strand on the intact matrix. In this intermediate, called a D-loop, the displaced strand would invade the homologous sequence of the other chromatid and the DNA would isomerize into a Holliday junction.

Based on the activity of Redα from the λ phage, which is a 5' to 3' exonuclease, Resnick proposed that the 3' ended tail produced was displaced to a homolog intact matrix, and served as primer for DNA polymerization, until the repair was long enough to be brought back to the initial break. The repair ended by a last DNA polymerization step. This is the first model of what is now called synthesis-dependent strand annealing (SDSA, Figure 11Eα, [START_REF] Resnick | The repair of double-strand breaks in DNA; a model involving recombination[END_REF]). However, this mechanism did not explain crossovers. So Resnick imagined further that after the 3' end invasion of an intact matrix, the endonuclease could nick the displaced DNA and promote crossover (Figure 11Eβ, [START_REF] Resnick | The repair of double-strand breaks in DNA; a model involving recombination[END_REF]). Following a different lane of thought, Szostak et al. proposed a model for crossing-overs based on the formation of double Holliday junction (Figure 11F, [START_REF] Szostak | The double-strand-break repair model for recombination[END_REF])): both 3' ends obtained after resection of a DSB invade a homologous sister chromatid and pair on it. After DNA synthesis and ligation, a double Holliday junction structure is obtained and can be resolved.

After that, the models have been inspired by previous model and did not evolve much. However, proteins capable of promoting these events were studied. The two strands of a broken chromosome fragment can form base pairs with an intact template and promote copying to the end of the template, thus producing a recombined, full-length product [START_REF] Meselson | Chromosome brekage accompanying genetic recombination in bacteriophage[END_REF]. B. Holliday's 1964 model. A pair of nonsister chromatids after meiotic DNA replication are shown; the two other chromatids, uninvolved in recombination, are not shown. A pair of same-strand nicks leads to a reciprocal exchange and formation of symmetric heteroduplex connected by a four-stranded symmetric structure now known as a Holliday junction (HJ). The HJ can be cleaved by cutting either of two pairs of strands (orientations 1 and 2). Crossovers occur when the HJ is cleaved so that only the crossing-strands connect the two homologous chromosomes. In the example shown, mismatch corrections lead to a 6:2 gene conversion [START_REF] Holliday | A mechanism for gene conversion in fungi[END_REF]. C. Charles Thomas's SSA model to obtain reciprocal recombination by annealing overlapping single strands of DNA from two chromosomes with offset nicks on both strands [START_REF] Thomas | Recombination of DNA molecules[END_REF]. D. Meselson and Radding's 1975 model. A single nicked strand is displaced by new DNA synthesis primed from the 3' end of the nick. The displaced strand can form a region of heteroduplex DNA by strand invasion and the formation of a displacement loop (D-loop). Cleavage of the D-loop leaves a single region of heteroduplex DNA adjacent to an HJ that is always distal from the initiating nick. Isomerization of the HJ and subsequent branch migration leads to the formation of a symmetric (sym. het) region of heteroduplex adjacent to an asymmetric (asym. het) segment (with only one heteroduplex region), still with the crossover point far from the initiating lesion [START_REF] Meselson | A general model for genetic recombination[END_REF]). E. The DSB repair model of [START_REF] Resnick | The repair of double-strand breaks in DNA; a model involving recombination[END_REF]. (α) A DSB is resected and one of the 3′ ends invades a template. The template strand is nicked. The paired DSB end initiates new DNA synthesis, which is displaced, allowing the original sequences to reanneal with the template. When the newly-copied strand is long enough, it can anneal with the second end, priming a second round of new synthesis and the repair of the DSB. This leads to a noncrossover repair of the DSB. (β) Crossovers can be generated by DSB repair. Here, the nicked template strand itself can anneal with the single-stranded sequences created by 5′ to 3′ resection. Strand invasion of the original DSB end creates a Holliday junction. An endonuclease nick at the base of the HJ results in connections between the molecules that result in a crossover. DNA synthesis fills in the ssDNA gaps and leads to a crossover [START_REF] Resnick | The repair of double-strand breaks in DNA; a model involving recombination[END_REF]). F. The double Holliday junction (dHJ) model of [START_REF] Szostak | The double-strand-break repair model for recombination[END_REF]. A DSB was enlarged into a double-stranded gapped region, which was subsequently resected to have 3′-ended single-stranded tails that could engage in strand invasion. The first strand invasion would produce a D-loop to which the end of the second resected end could anneal. The initial structure has one complete and one half HJ, but branch migration of the half HJ allows the formation of a second complete HJ. New DNA synthesis completes the formation of a fully ligated structure that can be resolved into crossovers if the two HJs are cleaved in different orientations. Resolution of both HJs as crossovers should leave an apparent double crossover in the middle; such outcomes are rare [START_REF] Szostak | The double-strand-break repair model for recombination[END_REF]. Adapted from [START_REF] Haber | RecA family proteins in archaea: RadA and its cousins[END_REF].

Lehman explored this activity. They tried to renaturate the NaOH-denatured 5386 bp long φX174 genome (circular duplex linearised by PstI digestion) in the presence of RecA and they demonstrated that when the RecA concentration is low, the protein needs ATP and Mg 2+ to promote the reaction.

They separated the products of renaturation on gel and observed them under an electronic microscope. A high molecular weight complex was separated on gels, and a network of DNA was seen under the microscope (Bryant, Lehman 1985). This suggests either that RecA promotes multiple single strand pairing reactions within the same ssDNA molecule, either it promotes another form of recombination: the strand invasion reaction. Indeed RecA is able to search homology by binding to ssDNA and searching of a complementary a dsDNA. This activity has been observed more precisely by single molecule assays (van der Heijden et al. 2008). A dsDNA is bound chemically to a surface at one end, and to a magnetic bead at the other end. A magnetic field pulls up the magnetic bead with a chosen force to elongate the DNA. The space position of the bead is measurable and represents the length of the DNA molecule. Homologous ssDNA covered of RecA is added thanks to a flow. Here the ssDNA can invade the dsDNA and pair to its complementary sequence. To verify the invasion the product is digested with a restriction enzyme. Without the strand invasion reaction, the digestion cuts the DNA and the bead is lost. But after the strand invasion, the digestion cut the dsDNA resulting of the annealing but the bead is retained by the displaced ssDNA strand. Furthermore, the author saw the structure after invasion is torsionally constrained by trying to turn the bead in both directions. This constraint is lost after the digestion. For the authors, this result indicates the displaced strand is wrapped around the heteroduplex dsDNA formed by the invasion.

Single molecule experiments have also permitted to ask questions about the search of homology by RecA. A first research looked for the sliding of RecA on DNA [START_REF] Ragunathan | RecA filament sliding on DNA facilitates homology search[END_REF]. To do so, a ssDNA labeled with an acceptor for fluorescence resonance energy transfer (FRET) is bound to a glass surface. RecA next covers the ssDNA. The target dsDNA is labeled with an acceptor for FRET. With this, the authors measured the distance between the acceptor and the donor as a function of time.

They found that RecA slides on dsDNA with a diffusion coefficient of 7700bp²/s until it finds homology with a minimum of 6 complementary nucleotides. This rapid 1D search is to be confronted with another discovery where the authors observed that the ssDNA-RecA complex can be transfered from a dsDNA segment to another (Forget, Kowalczykowski 2012). A dsDNA is bound between two beads, and the distance between them can be controlled, thereby changing the containment of the DNA. They showed that the less the DNA is constrained and the longer the homology, the quicker is the reaction. They also showed that ssDNA-RecA complexes can create a bridge inside the dsDNA to search homology at two different positions at the same time with two different part of the ssDNA. The question now is how RecA finds this homology. For this, the resolution of the structure of RecA has been very suggestive. Story et al. resolved its structure in 1992. This protein possesses a core Nterminus (Nt) domain and a smaller C-terminus (Ct) domain. The core-domain has two activities: it binds DNA and binds and hydrolyzes ATP. The Ct domain is a secondary DNA binding domain. RecA polymerizes on DNA in a helix formation, with 6 monomers of RecA per turn. The core DNA binding site is at the center of the structure, but the Ct one is turned to the outside [START_REF] Story | The structure of the E. coli recA protein monomer and polymer[END_REF]. With this structure, De Vlaminch et al. (2012) proposed a mechanism for homology recognition by a shift of DNA between the two binding sites. With a single molecule set-up of bead and dsDNA as described previously, they tried to make the DNA move with a DNA-RecA complex (Figure 12). They found that the secondary DNA binding site of RecA can bind to ssDNA but not to dsDNA, unlike the primary DNA binding site, which can bind both. However the secondary DNA binding site can bind dsDNA in negative supercoiled structures, suggesting it can bind to an unwinding DNA. This suggests the homology search begins in a breathing DNA. One of the two strands of the breathing DNA is caught by the secondary DNA binding site of a RecA-ssDNA complex, and then the homology is found on the primary DNA binding site (De Vlaminck et al. 2012). The ATPase site of RecA permits the dissociation of the RecA-DNA complex. Once the homology is found, the RecA filament continues to polymerize to continue the annealing [START_REF] Rossi | The RecA/RAD51 protein drives migration of Holliday junctions via polymerization on DNA[END_REF]. By this mechanism, there is the creation a Holliday junction. This junction is next resolved by dissolution of the annealing or by cleavage into the junction.

In vivo the question is a little different. Indeed RecA is used to repair breaks into the chromosome of bacteria. Nevertheless, how can RecA find homology between two DNA segregated at the two poles of the cell in preparation for the division? DNA damage can occur long times after the DNA polymerization, so we need to imagine how the two DNA are caught and brought together. This question has been addressed in the laboratory of Pr. Sherratt [START_REF] Lesterlin | RecA bundles mediate homology pairing between distant sisters during DNA break repair[END_REF]). In a system creating double strand breaks that are detectable under the optic microscope, they used a GFP tagged RecA to observe how the RecA-DNA complex acts at the breaking point. They observed that RecA is able to displace the break cross the cell to find homology on the sister DNA. After the repair, the two sister DNAs are separated across the cell in prevision of the cellular division.

B) Regulation of the RecA filament

Regulation for the RecA filament occurs at two distinct steps. The first one is the regulation before filament formation, which is inhibited by SSB and promoted by RecBCD or RecFOR. The second is the regulation after filament formation, stabilized by RecX and disassembled by DinI, UvrD and maybe by RdgC (Figure 13).

It is usually admitted that SSB inhibits the RecA filament formation by binding the ssDNA before RecA. Indeed in vitro when both proteins are mixed together, SSB catches the ssDNA before RecA [START_REF] Reddy | Binding of SSB and RecA protein to DNAcontaining stem loop structures: SSB ensures the polarity of RecA polymerization on singlestranded DNA[END_REF]. Nevertheless, if RecA is already nucleated on the ssDNA, SSB facilitates the polymerization of RecA by unwinding the secondary structures of ssDNA. A recent single molecule study (Bell et al. 2012) showed that RecA is able to nucleate on an ssDNA covered by SSB but with a high lag time (3 minutes) and a low nucleation rate. However, the RecA filament polymerization is not disturbed.

Chapter I: Introduction, Homologous recombination, RecA 37 RecA has manners to overcome the SSB barrier. The first one relies on RecBCD activity. This complex is composed of three different proteins: RecB, that has helicase and nuclease activities, RecC, which recognizes a specific site on DNA called Chi, and RecD, which is an helicase. This heterotrimer detects DSB on DNA and degrades both strands until RecC recognizes a Chi site. RecBCD pauses on Chi sites Figure 13: Regulation of RecA activity. There are two RecA loading pathways depending on the DNA damage. In the case of double strand break (DSB), RecBCD recognize blunt DNA (1, RecB in blue, RecC in Yellow, RecD in green) and degrade both strand but not at the same speed (2). When RecB recognize a χ site, the 3' to 5' exonuclease activity stop but not the 5' to 3' and RecC load RecA on the 3' overhang created (3). The ssDNA-RecA filament complex is ready to recombine (4). In the other case of single strand break (SB), the DNA is digested and SSB load into the ssDNA produced (5). RecF recognise a ssDNA break [START_REF] Baker | Recombination and modular exchange in the genesis of new lambdoid phages[END_REF] and load RecOR on the ssDNA (7, RecO in brown and RecR in grey). RecOR moves SSB away from the ssDNA permiting the loading of RecA [START_REF] Muylkens | Coinfection with two closely related alphaherpesviruses results in a highly diversified recombination mosaic displaying negative genetic interference[END_REF]. The ssDNA-RecA filament complex is ready to recombine [START_REF] Hatfull | Bacteriophage genomics[END_REF]. RecX can dismantle the RecA filament [START_REF] Niwa | Sequence homology in DNA molecules of temperate phages phi81, phi80 and lambda[END_REF] and DinI stabilise it [START_REF] Hendrix | Bacteriophage genomics[END_REF]. Each one are interchangeable [START_REF] Canchaya | Prophage genomics[END_REF]. If the RecA filament remain stable [START_REF] Botstein | A theory of modular evolution for bacteriophages[END_REF], it searches for homology into an intact matrix by strand invasion [START_REF] Lawrence | Imbroglios of viral taxonomy: genetic exchange and failings of phenetic approaches[END_REF]. and its activity changes to degrade only one of both strands. RecB loads RecA on the 3' overhang DNA created before SSB come [START_REF] Spies | The RecA binding locus of RecBCD is a general domain for recruitment of DNA strand exchange proteins[END_REF]).

The second one is the RecFOR pathway. RecF recognizes a single strand break on dsDNA. RecO can bind to ssDNA, RecF, RecR and SSB. A study showed it also anneals two complementary ssDNA, in the presence and in the absence of SSB [START_REF] Kantake | Escherichia coli RecO protein anneals ssDNA complexed with its cognate ssDNA-binding protein: A common step in genetic recombination[END_REF]. RecR binds a dimer RecO. The idea here is that the RecOR complex either binds to SSB and moves it away to permit the nucleation of RecA, or binds to RecF which finds a ssDNA break on dsDNA and polymerizes RecA from the break (Morimatsu, Kowalczykowski 2003;Handa et al. 2009). Recent single molecule analyses showed that RecOR and RecFOR complexes decrease the lag of RecA nucleation and increase the polymeration rate (Bell et al. 2012). Last years, the structure of RecOR was resolved [START_REF] Radzimanowski | An 'open' structure of the RecOR complex supports ssDNA binding within the core of the complex[END_REF] and revealed a ring structure composed by two RecO and four RecR. This ring rounds the ssDNA that permits to push SSB and nucleate RecA.

RecX and DinI have antagonist activities. RecX is expressed in operon with RecA and has an inhibition activity. RecX binds to RecA and inhibits its polymerization, leaving it to its depolymerization, whereas DinI binds to RecA, and stabilizes the RecA filament preventing the depolymerization and promoting the polymerization [START_REF] Lusetti | The DinI and RecX proteins are competing modulators of RecA function[END_REF][START_REF] Renzette | DinI and RecX modulate RecA-DNA structures in Escherichia coli K-12[END_REF].

UvrD is a helicase which dismantles the RecA filament (Veaute et al. 2005). UvrD prevents the polymerization of RecA at the replication fork in vivo. This may prevent the reannealing of the fork by RecA [START_REF] Lestini | UvrD controls the access of recombination proteins to blocked replication forks[END_REF].

RdgC is the most ambiguous inhibitor protein. Some activities were shown in vitro, but no phenotypes were observed in vivo. Indeed, when RecA is competed by RdgC in vitro in the classic strand exchange reaction, RecA loses its activity proportionally to RdgC addition [START_REF] Drees | Inhibition of RecA protein function by the RdgC protein from Escherichia coli[END_REF].

The structure was resolved in 2007 [START_REF] Briggs | Ring structure of the Escherichia coli DNA-binding protein RdgC associated with recombination and replication fork repair[END_REF]. It dimerizes to form a ring that rounds the DNA with a preference for the ssDNA. The idea is that RdgC slides on DNA and moves away RecA from DNA to inhibit its action.

C) Fidelity of this system

It is admitted that homologous recombination is a very faithful mechanism. However, it is currently discussed. Malkov et al. studied this property using a strand invasion assay, whereby a supercoiled plasmid is incubated with a labeled short oligonucleotide having more or less homology with the plasmid. If RecA pairs the oligonucleotide to the plasmid, a D-loop is formed and easily separated on gel. Using a 27-mer oligonucleotides, with 6 nt of perfect identity on both extremities with the plasmid, and 15 nt of random sequence in the center, they showed that RecA can recombine until diverged sequences 4 mismatches on 15bp (~25% of divergence) randomly distributed but in their model, mismatches were surrounded by short perfect homologies (6bp, (Malkov, Sastry, Camerini-Otero 1997)). Sagi et al. have proposed a molecular model for RecA fidelity: RecA may have a low sensibility to mismatches in the absence of ATP hydrolysis. This in vitro situation is nevertheless quite unlikely in vivo, unless the bacterium is running out of ATP. It succeeds to anneal strands more rapidly if the first mismatch is distant from the invading 3' end. The sensitivity to mismatches and the directionality in the strand exchange process support a mechanism for homology recognition that can be modeled as a kinetic proofreading cascade: RecA pairs the DNA monomer by monomer until the loss of similarity, thus RecA is stopped by consecutive mismatches [START_REF] Sagi | High fidelity of RecA-catalyzed recombination: a watchdog of genetic diversity[END_REF].

Beyond the intrinsic sensitivity of RecA to mismatches, a second mechanism for proofreading relies on the mismatch repair of the cell. MutS is the sensor of this system by detecting mismatches, with different affinity, depending on the mismatch (Brown, Brown, Fox 2001). MutL binds to MutS and polymerises on DNA. MutS recognizes a mismatch during the recombination exchange and together with MutL blocks the recombination intermediate by preventing rotation of DNA strands during the strand exchange. UvrD comes next to disrupt the RecA filament. Consequently, the invading strand is removed (Tham et al. 2013).

D) Links with Eukaryotes and Archaea

Proteins sharing homology with the Bacterial RecA are found in the two other branches of life. In Eukaryotes, Rad51 has the same function as RecA, although the homology spans only the core domain, that is an ATPase and a DNA binding domain. As already explained, RecA has a Ct domain that binds ssDNA. In Rad51, this is a Nt domain that has this function, sharing no homology with the Ct of RecA. Eukaryotes have several other Rad51 paralogs, which will not be discussed here. In Archaea, RadA is the ortholog of Rad51, having the same Nt domain. Furthermore, Archaea have a paralog of RadA called RadB. Lin et al. tried to reconstitute the evolution history of these genes (Figure 14 [START_REF] Lin | Origins and evolution of the recA/RAD51 gene family: evidence for ancient gene duplication and endosymbiotic gene transfer[END_REF]). They compared sequences of Rad51 and its paralogs from different Eukaryota, RadA and RadB from Archaea, and RecA from Bacteria and plants. They think that the RecA ancestor was conserved in the reign of Bacteria. In the common ancestor of Eukaryotes and Archaea, there was a gene, or genome, duplication, leading to two copies of the recA gene, that they RecA and Rad51 were compared for their in vitro activities. Both promote DNA pairing and strand exchange. They both need ATP and magnesium to do so (Rice et al. 2001). RadA and RecA were also compared. In the same manner RadA promotes ATP hydrolysis, DNA pairing and strand exchange (Seitz et al. 1998). Recently, Rad51 was spoted by chromatin immunoprecipitation in vivo on yeast genome with a system of inducible DSB. Rad51 binds first to the DSB as expected, and on the homologous region of the DSB [START_REF] Renkawitz | Monitoring homology search during DNA double-strand break repair in vivo[END_REF]. In yeasts and mammals, paralogs are used to stabilize the Rad51 filament (see [START_REF] Karpenshif | From yeast to mammals: recent advances in genetic control of homologous recombination[END_REF] for review). In Archaea, RadB also enhances the activity of RadA strand invasion [START_REF] Graham | An archaeal RadA paralog influences presynaptic filament formation[END_REF].

3) Phage recombinases

As said earlier, three families of recombinases are encoded by phages (Lopes et al. 2010). Each family has its particularities and its model of study. Here we propose to present recent researches on these different models to illustrate the capacities of each family. The gene duplication that occurred before the divergence of archaea and eukaryotes gave rise to two lineages, RADα and RADβ, and, in eubacteria, recA has remained as a single-copy gene. In eukaryotes, both RADα and RADβ genes experienced several duplication events, but, in archaea, they remained as single-copy genes. Eukaryotic recA genes originated from proteobacteria (recAmt) and cyanobacteria (recAcp) recA genes after two separate endosymbiotic events. recAmts were subsequently lost in the ancestors of animals and fungi [START_REF] Lin | Origins and evolution of the recA/RAD51 gene family: evidence for ancient gene duplication and endosymbiotic gene transfer[END_REF].

A) Rad52-like recombinases

The most studied Rad52-like recombinase is Redβ from λ. This protein is known to promote SSA but not strand invasion (SI, Figure 11). In vivo, SSA is the observed way of action for Redβ (Stahl et al. 1997). However, when Redβ has annealed a part of a ssDNA on its complementary ssDNA, it can promote branch migration along the dsDNA until the end of the complementarity region [START_REF] Li | The beta protein of phage lambda promotes strand exchange[END_REF]. In vitro, Redβ can promote SI under conditions of low Mg 2+ concentration (0 to 2 mM) (Rybalchenko et al. 2004). Another well studied Rad52-like recombinase, the RecT encoded by the rac E. coli prophage, has the same capacity at low magnesium concentration (Noirot, Kolodner 1998).

In fact, under such conditions, the dsDNA is less stable, because the negative charges of phosphates are not compensated by the positive charges of the magnesium. The two strands of the dsDNA repel themselves and open the double strand. The complementary ssDNA covered by Redβ can anneal into this breathing dsDNA. In vivo, the supercoiled dsDNA breathes too. However, for strand invasion to proceed, the breathing needs to be at the exact position where the ssDNA caught by Redβ can anneal, and it is a rare event. After that, Redβ can promote the branch migration and complete the reaction for homologous recombination.

Redβ has not been crystallized yet, but electron microscopy observations have permitted to observe its structure. It forms ring structures. In the absence of DNA, the ring is composed of 12 subunits, whereas with ssDNA, it is made of 15 to 18 subunits. The structure changes in the presence of dsDNA: Redβ becomes a helical filament (Figure 15A, [START_REF] Passy | Rings and filaments of beta protein from bacteriophage lambda suggest a superfamily of recombination proteins[END_REF]). The hypothesis is that in the ring structure form, Redβ exposes ssDNA at the outside of the ring. After annealing, Redβ protects the dsDNA inside the helical filament. The biochemical behavior of Redβ was reminiscent of that of yeast Rad52, which forms also such rings under the microscope, but no homology was found, even by the Koonin group many years later (Iyer, Koonin, Aravind 2002) proposed a different mechanism of SSA, where the λ exonuclease, Redα, digests completely one strand of the dsDNA. Redβ anneals the extremities of this new ssDNA at the replication fork (Figure 16, (Maresca et al. 2010)). This annealed DNA is then used as a primer for an Okazaki fragment.

Furthermore, if the DnaG primase is mutated so that less primers are formed on the lagging strand template, the recombination is enhanced [START_REF] Lajoie | Manipulating replisome dynamics to enhance lambda Red-mediated multiplex genome engineering[END_REF]. A recent study showed that Pol III, the major DNA polymerase at the replication fork, had a strong effect on dsDNA integration, but a low effect on ssDNA integration at the replication fork [START_REF] Poteete | Involvement of DNA Replication Proteins in Phage Lambda Red-Mediated Homologous Recombination[END_REF]. In summary, all present models for recombination by Redβ include an annealing step at the replication fork.

The fidelity of Redβ is much lower than that of RecA. In 2008, Martinsohn et al. found that Redβ recombined inverted 800 bp long sequences, up to 22% of divergence, with an efficiency 100 fold higher than RecA (Martinsohn, Radman, Petit 2008). A recent study (Li et al. 2013) also showed that Redβ recombined diverged sequences up to 17% using a different assay, the annealing of an oligonucleotide at the replication fork (also named single-strand recombineering). In addition, they found that the position of mismatches had impact on the recombination frequency: the most important for insertion of an oligonucleotide is the complementarity at the two extremities of the oligonucleotide, the 5' extremity permits the annealing before the mutation point and the 3' extremity permits the loading of the polymerase to complete the insertion. The authors also showed that the polymerases Pol I and III have major impact on the recombination frequency in the recombineering reaction.

The Eukaryotic homologue of Redβ is named Rad52. Its main function in yeast is to mediate Rad51 binding to DNA, in a way analogous to RecBCD or RecFOR in bacteria. This function of Rad52 is essentially carried out by its Ct domain, which contains Rad51-interacting sequences. This domain is absent in phage homologs. In addition, yeast Rad52 performs Rad51-independent recombination in vivo, by an SSA mechanism [START_REF] Mortensen | DNA strand annealing is promoted by the yeast Rad52 protein[END_REF]. This activity lies in its Nt domain, which is homologous to the phage proteins, and has been crystallized. This SSA activity has also been demonstrated in vitro [START_REF] Van Dyck | Visualization of recombination intermediates produced by RAD52-mediated single-strand annealing[END_REF]). In addition, Rad52 promotes strand invasion in a supercoiled dsDNA, and D-loops can be observed on gel [START_REF] Kagawa | Homologous pairing promoted by the human Rad52 protein[END_REF]. Inspired by the fact that Rad52 is an accessory protein for Rad51 homologous recombination, in vitro experiments have shown that the RecA strand exchange activity is stimulated by the phage-encoded Sak recombinase (Ploquin et al. 2008), which shares a detectable homology to Rad52, (Iyer, Koonin, Aravind 2002).

However, no in vivo evidence strengthens this set of data. On the contrary, for Redβ, we have shown that its recombination activity in vivo is independent of RecA (see chapter II). Interestingly, it was recently recognized that yeast encode a short Nt version of the Rad52 protein, called Mgm101, exported into the mitochondria that promotes SSA and has the same ring structure under electron microscope. This protein is essential for DNA repair in mitochondria [START_REF] Mbantenkhu | Mgm101 is a Rad52-related protein required for mitochondrial DNA recombination[END_REF]).

B) Gp2.5-like recombinases (a) On phages

Gp2.5 is an SSB-like protein encoded by the phage T7 infecting E. coli. It was first shown to bind ssDNA and to be needed for T7 DNA replication [START_REF] Kim | Purification and characterization of the bacteriophage T7 gene 2.5 protein. A single-stranded DNA-binding protein[END_REF]). Later, it was observed that it anneals two ssDNA without hydrolyzing ATP. Finally, with a classic test of strand exchange, it was demonstrated that Gp2.5 promotes the formation of joint molecules [START_REF] Kong | Single-stranded DNA binding protein and DNA helicase of bacteriophage T7 mediate homologous DNA strand exchange[END_REF], provided that the T7 helicase Gp4 is added to the strand exchange reaction. It may be that Gp2.5 is not able to promote branch migration alone, but helped with an helicase it can do so. Hollis et al. crystallized Gp2.5 in 2001. It has the classical OB-fold found in SSB protein with a supplementary α-helix, positioned in the sequence between the β-sheets forming the OB-barrel. This structure suggests that Gp2.5 binds to ssDNA as a monomer, unlike SSB that binds ssDNA as dimers.

It is possible that the α-helix plays a crucial role in the annealing activity (Hollis et al. 2001).

(b) The ICP8 orthologs in Herpes Simplex Virus ICP8 from HSV (Herpes Simplex Virus) and Gp2.5 from T7 share a common ancestor [START_REF] Kazlauskas | Two distinct SSB protein families in nucleo-cytoplasmic large DNA viruses[END_REF]. Indeed, different ICP8liks and Gp2.5-like were aligned by profile comparison. The core of ICP8 revealed the same structure as Gp2.5. These two proteins are known to have a function related to replication and recombination.

A ring structure in the presence of ssDNA was observed by electron microscopy for ICP8 [START_REF] Tolun | Details of ssDNA annealing revealed by an HSV-1 ICP8-ssDNA binary complex[END_REF]. Two superposed homononamer rings compose this structure. The authors fitted the crystal structure into their reconstruction map. They hypothesize that the ssDNA is inside the ring structure and each two sub-rings present ssDNA to the other (Figure 17). From here, they made a novel SSA model where the two rings slide one on another until they matched two complementary ssDNA.

C) Rad51-like recombinases

Rad51-like recombinases can be split into two sub-families: RecA orthologs, named UvsX, and coreonly RecA, named Sak4.

(a) UvsX

UvsX is a protein encoded by the phage T4. The UvsX structure was resolved in 2011. It is strikingly similar to RecA [START_REF] Gajewski | Crystal structure of the phage T4 recombinase UvsX and its functional interaction with the T4 SF2 helicase UvsW[END_REF]. UvsX has also the same activity as RecA: it promotes strand invasion and branch migration, in an ATP-dependent manner. UvsX works better and quicker to promote strand exchange in the presence of the phage helicase UvsW. A recent review compared UvsX, RecA and their homologues in yeast and human, RAD51. It appears that UvsX acts quicker than the others do, in the ATPase activity and strand exchange activity (Liu et al. 2011). However, RAD51 (C) The UvsY recombination mediator protein forms a tripartite complex with Gp32 and ssDNA and "primes" the complex for recruitment of UvsX recombinase. (D) UvsY recruits ATP-bound UvsX protein and nucleates presynaptic filament formation. Gp32 is displaced in the process. (Liu et al. 2011) and homologous ssDNA. This correlates with the present model for recombination: UvsX binds to ssDNA and searches for homology into dsDNA. After the resolution of the recombination intermediate, UvsX releases the dsDNA by hydrolyzing ATP [START_REF] Maher | Coordinated Binding of Single-Stranded and Double-Stranded DNA by UvsX Recombinase[END_REF].

(b) Sak4

The Sak4 family was discovered with three other protein families called Sak, Sak2 and Sak3 in an in vivo assay (Bouchard, Moineau 2004). Lactoccoccus lactis possesses systems that stop the phage infection. They are called Abi for "abortive infection". The authors searched for mutations in phages that escape to the AbiK system. They classified these mutations into four families, named above. Sak, Sak2 and Sak3 were identified to be recombinases of the Rad52 family. But Sak4 remained different from the three first, and was supposed to be a recombinase only by analogy. In 2010, Lopes et al. showed that Sak4 is a core-only RecA which promotes SSA in vivo (recombineering assay, (Lopes et al. 2010)). Sak4 was not further studied at that time, but some other core-only RecA had been studied in various living organisms, which are briefly presented below.

In Eukaryotes, Rad51 paralogs help Rad51 to promote homologous recombination. First in S. cerevisiae, two core-only RecA, Csm2 and Psy3, and two other proteins, Shu1 and Shu2, compose the SHU-complex. This complex permits the polymerization of the Rad51 filament and stabilizes it [START_REF] She | Structural and SAXS analysis of the budding yeast SHU-complex proteins[END_REF]). In human, the complex BCDX2 has a core-only RecA designated Xrcc2. The other proteins of this complex, Rad51B, Rad51C and Rad51D are Rad51 paralogs with the same core-domain and with different Nt domain. This complex promotes SSA, and binds to branched-DNA (Yokoyama et al. 2004). In vivo, this complex participates to the recruitment of Rad51 on ssDNA [START_REF] Chun | Rad51 paralog complexes BCDX2 and CX3 act at different stages in the BRCA1-BRCA2-dependent homologous recombination pathway[END_REF].

In Archaea, two RadA paralogs are core-only RecA: RadB and RadC. RadB is only found in Euryarchaea. It binds ssDNA and dsDNA with the same efficiency as RadA. RadB binds and hydrolyzes ATP but does not release ADP. It does not promote strand exchange either and inhibits the strand exchange activity of RadA when both are mixed together. However, when RadB is added 15 minutes after RadA in a strand exchange test, the RadA activity is improved (Komori et al. 2000). In this study, RadB seems to regulate the Holliday junction clivage by an inhibition in the absence of ATP.

RadC was first annotated as KaiC because of its high level of homology with KaiC, the circadian clock protein found in Cyanobacteria. The only difference is that KaiC had two RecA core domains while RadC has one only [START_REF] Haldenby | RecA family proteins in archaea: RadA and its cousins[END_REF]. Deletions in the radC gene provoke sensitivity to DNA damage agents. So these genes participate to the repair of DNA damages, contrary to kaiC genes [START_REF] Liang | Knockouts of RecA-like proteins RadC1 and RadC2 have distinct responses to DNA damage agents in Sulfolobus islandicus[END_REF].

Chapter I: Introduction, All the questionings, Phage recombinases 48

IV) All the questionings

The mechanisms permitting the remarkable acquisition of mosaic structures for temperate genomes are unknown at present. In our team, we think the unique activities of phage recombinases participate to this acquisition. We are particularly interested by their capacity to recombine diverged sequences, unlike RecA. The Martinsohn et al. study had clearly suggested a higher performance of Redβ on 22% diverged sequences, compared to RecA (Martinsohn, Radman, Petit 2008). However, the complete demonstration that this recombination was Redβ dependent could not be obtained, because the recombination assay itself relied on Redβ expression for the read-out. This led us to set up a different assay, where the acquisition of a chromosomal gene marker by the phage is followed up. Depending on the presence of homologous regions flanking this marker, we found its efficient acquisition by the phage. The question was to know if these exchanges of genetical material are mediated only by phage recombinases, only by the host recombinase RecA or both. The result is to be confronted against the hypothesis of Bobay et al., where the phage needs to choose between a phage homologous recombination system and this of the host (Bobay, Rocha, Touchon 2013). This first work is reported in chapter II. My particular contribution to this work has been to address the generality of the observation made with Redβ. Have other Rad52-like recombinases relaxed fidelity as well? I also investigated phage genomes with a bio-informatics approach, to systematically detect recent exchanges and scan for the presence of traces of homologous recombination at their vicinity.

In a second period of my research, I decided to turn to the study of Sak4 recombinases, as these were newly uncovered, and poorly characterized. I used a combined genetics and biochemical approach, to characterize this core-only RecA, and find out whether it could recombine DNA by itself, or would rather behave as accessory factor, as found in Eukaryotes and Archaea. Would this recombinase promote SSA like phage recombinases of the Rad52 family, or SI like UvsX and RecA?

And would its fidelity be more similar to RecA-like recombinases, or to the Rad52-like recombinases I had studied first? This is reported in chapter III of this Manuscript.

Introduction

Bacteriophages, or phages, the viruses that attack bacteria, have gained a renewed interest in the last decade with the emergence of antibiotic resistant bacteria. Despite their early discovery [1,2] and the in-depth molecular and genetic characterization of few model phages, overall phage biology is still globally poorly understood comparatively to their ecological importance. Indeed, phages and related elements dominate the biosphere both numerically and in terms of genetic diversity. Despite more than 10 5 genes already described in phage genomes, recent studies suggest that the majority of phage genes remains to be discovered [3]. The great genetic diversity of these viruses is due to their very ancient origin, their large population size and their high evolvability. Understanding evolvability of bacterial viruses will likely become a major issue with the prospective massive use of phages as alternatives to antibiotics. Notably, the mutation rate of viruses is much higher than that of cellular organisms [4,5]. Horizontal gene transfer also plays a major role in virus evolution by creating new combinations of genetic material through the pairing and shuffling of related DNA sequences [START_REF] Baker | Recombination and modular exchange in the genesis of new lambdoid phages[END_REF][START_REF] Van Der | Rapid host adaptation by extensive recombination[END_REF][START_REF] Muylkens | Coinfection with two closely related alphaherpesviruses results in a highly diversified recombination mosaic displaying negative genetic interference[END_REF].

Initial observations of hybridizing segments between phage genomes by electron microscopy, and more recent genomic analyses, have revealed the pervasive mosaicism of temperate phage genomes [START_REF] Hatfull | Bacteriophage genomics[END_REF][START_REF] Niwa | Sequence homology in DNA molecules of temperate phages phi81, phi80 and lambda[END_REF][START_REF] Hendrix | Bacteriophage genomics[END_REF]. Mosaicism refers to the patchwork character of phage genomes, which can be considered as unique combinations of exchangeable genomic segments [START_REF] Hendrix | Bacteriophage genomics[END_REF][START_REF] Canchaya | Prophage genomics[END_REF][START_REF] Botstein | A theory of modular evolution for bacteriophages[END_REF][START_REF] Lawrence | Imbroglios of viral taxonomy: genetic exchange and failings of phenetic approaches[END_REF][START_REF] Brussow | Comparative phage genomics and the evolution of Siphoviridae: insights from dairy phages[END_REF]. Temperate phages, as opposed to lytic phages, have the ability to enter a prophage dormant state upon infection, in which they stably replicate with the bacterial genome. Nearly all bacterial genomes contain multiple active or defective prophages, the latter being unable to produce phage particles. In Escherichia coli, prophage genes can constitute up to 14% of the genome [START_REF] Hayashi | Complete genome sequence of enterohemorrhagic Escherichia coli O157:H7 and genomic comparison with a laboratory strain K-12[END_REF], and represent 41% of a 20 species pangenome [START_REF] Hayashi | Complete genome sequence of enterohemorrhagic Escherichia coli O157:H7 and genomic comparison with a laboratory strain K-12[END_REF][START_REF] Bobay | The adaptation of temperate bacteriophages to their host genomes[END_REF][START_REF] Tenaillon | The population genetics of commensal Escherichia coli[END_REF]. Intergenomic rearrangements are thus facilitated for temperate phages by frequent encounters of different viruses inside the same bacterial host, for example between an invasive virus and a resident prophage.

While genome shuffling appears as a key driver of phage evolution, a quantitative description of mosaicism and analysis of its underlying molecular mechanisms are lacking. In particular, it is still debated whether phage genetic mosaicism is the product of recombination at sites of limited homology between genomes [START_REF] Martinsohn | The lambda red proteins promote efficient recombination between diverged sequences: implications for bacteriophage genome mosaicism[END_REF][START_REF] Clark | Nucleotide sequence of coliphage HK620 and the evolution of lambdoid phages[END_REF], or the result of random, cut and paste, illegitimate recombination [START_REF] Hendrix | Bacteriophage genomics[END_REF][START_REF] Hatfull | Bacteriophages and their genomes[END_REF]. In the latter hypothesis, the conservation of synteny would result from the counterselection of deleterious non-ordered gene combinations. Functions involved in homologous recombination (HR) have been extensively studied in E. coli and phage l [START_REF] Murphy | Phage recombinases and their applications[END_REF][START_REF] Kuzminov | Recombinational repair of DNA damage in Escherichia coli and bacteriophage lambda[END_REF]. In E. coli, the RecA recombinase is essential for catalyzing DNA exchanges between homologous molecules. However, the rate of successful RecA-dependent exchanges rapidly decreases with increasing sequence divergence [START_REF] Shen | Homologous recombination in Escherichia coli: dependence on substrate length and homology[END_REF][START_REF] Majewski | DNA sequence similarity requirements for interspecific recombination in Bacillus[END_REF], suggesting that homologous exchanges should not happen between very divergent phage genomes. Phage genomes also encode recombinases catalyzing HR reactions, that have been classified into 3 super-families known as Rad51-, Rad52-and Gp2.5-like [START_REF] Lopes | Detection of novel recombinases in bacteriophage genomes unveils Rad52, Rad51 and Gp2.5 remote homologs[END_REF]. These recombinases are also found sporadically on nonbacterial viruses: archaeal proviruses encode Rad52-like genes [START_REF] Duboc | Connecting dysbiosis, bile-acid dysmetabolism and gut inflammation in inflammatory bowel diseases[END_REF] and Mimi and Herpes viruses encode a recombinase sharing homology with Gp2.5 [START_REF] Turpin | Behavior of lactobacilli isolated from fermented slurry (ben-saalga) in gnotobiotic rats[END_REF]. Among the phage recombinases, the Rad52-like family is the largest and most diversified, and is itself subdivided into the Redb, Erf and Sak groups. Among them, the l recombinase Redb is known to be efficient in the recombination of diverged sequences [START_REF] Martinsohn | The lambda red proteins promote efficient recombination between diverged sequences: implications for bacteriophage genome mosaicism[END_REF][START_REF] Li | Bacterial DNA polymerases participate in oligonucleotide recombination[END_REF], leading us to suggest that phage recombinases could be key actors in genomic shuffling of related phages.

The l HR system, known as Red, is expressed during phage lytic development. Red consists of two functions encoded by the redb and reda genes. The main activity of Redb is to mediate singlestrand DNA annealing between a Redb-bound single-stranded region and a complementary sequence. l Reda is a double-strandspecific exonuclease that generates single-strand DNA for Redb annealing. Redb can also promote recombination by a RecA-like strand-invasion mechanism, especially on short DNA sequences [START_REF] Muyrers | RecE/RecT and Redalpha/Redbeta initiate double-stranded break repair by specifically interacting with their respective partners[END_REF]. Two other genes in the l nin genomic region, orf (former name ninB) and rap (former name ninG), were shown to facilitate RecA-dependent gene exchanges in vivo between strictly identical sequences [START_REF] Hayes | NinR-and red-mediated phageprophage marker rescue recombination in Escherichia coli: recovery of a nonhomologous immlambda DNA segment by infecting lambdaimm434 phages[END_REF]. The orf gene product is a mediator protein that participates in the loading of the bacterial RecA recombinase on SSB-coated DNA in the absence of the three bacterial proteins RecFOR [START_REF] Curtis | The Cterminus of the phage lambda Orf recombinase is involved in DNA binding[END_REF][START_REF] Sawitzke | Roles for lambda Orf and Escherichia coli RecO, RecR and RecF in lambda recombination[END_REF][START_REF] Tarkowski | Gene products encoded in the ninR region of phage lambda participate in Red-mediated recombination[END_REF][START_REF] Sharples | Holliday junction binding and resolution by the Rap structure-specific endonuclease of phage lambda[END_REF][START_REF] Poteete | Modulation of DNA repair and recombination by the bacteriophage lambda Orf function in Escherichia coli K-12[END_REF]. The rap gene codes for a Holliday junction resolvase [START_REF] Sharples | Holliday junction binding and resolution by the Rap structure-specific endonuclease of phage lambda[END_REF]. Orf and Rap have numerous homologs among temperate phages, and form ,500 members families in Pfam. Interestingly, their distribution pattern among phage genomes is contrasted: among the 465 completely sequenced phage genomes collected in the ACLAME database, 191 encode a recombinase [START_REF] Lopes | Detection of novel recombinases in bacteriophage genomes unveils Rad52, Rad51 and Gp2.5 remote homologs[END_REF]. The presence of Orf is tightly associated with Recombinase+ genomes, as among the 55 phages encoding Orf, 49 also encode a recombinase, of the Rad52-or Rad51-like family. On the contrary, among the 180 genomes encoding Rap, only 100 are Recombinase+, as if Rap and phage recombinase occurrences were independent (MAP et al., to be published elsewhere). These distributions are suggestive of a more important role of Orf on phage recombinase activity. However, whether Orf or Rap stimulates Redb mediated recombination is unknown at present.

Here, we study quantitatively the generation of mosaics between functional (i.e., infectious) temperate phages and defective prophages, and identify the genetic determinants of these exchanges. These assays reveal the preponderant role of Rad52like phage recombination genes in exchanges involving short and diverged sequences. Moreover, a global analysis of mosaics between active temperate phages and defective prophages further reveals that these exchanges are commonplace in phage genome evolution.

Results

A recombination assay to measure formation of mosaics in phage l

The aim of this first assay was to determine the extent and the mechanisms of genomic exchanges between an invasive infectious phage and defective prophages residing in the host chromosome. Homologous genomic regions between the MG1655 E. coli strain and l phage were identified by a Blast search using relaxed parameters (same parameters as for the ANI analysis, see Fig. S6 legend). Twelve regions sharing over 70% mean identity on a stretch of at least 100 bp were found, all inside defective prophages. 3 such regions, differing in size and in the extent of identity, were selected for the experiments: i) a region in Dlp12 defective prophage, sharing 98% mean identity over 3353 bp with l, ii) a region in Qin defective prophage sharing 96% mean identity over 657 bp, and iii) a region in Rac defective prophage sharing 88% mean identity over 703 bp, followed by another homology region nearby of 95% mean identity (Fig. 1 A, B &C). The length distribution of segments of perfect identity (segment without mismatch) is accordingly very different in the 3 regions (Fig. S1). Notably, the longest stretches of perfect identity are 298, 115 and 49 bp for Dlp12, Qin and Rac respectively. An antibiotic resistance gene, conferring resistance to either chloramphenicol (cat) or kanamycin (kanR), was introduced in the middle of each selected region (Fig. 1 and Materials and Methods). As a control, we used a strain with the cat gene into ilvD, which has no homology with l and whose deletion does not impact phage growth.

Defective prophages can reportedly excise and even replicate in different strain backgrounds under certain conditions, notably during phage infection [START_REF] Hayes | NinR-and red-mediated phageprophage marker rescue recombination in Escherichia coli: recovery of a nonhomologous immlambda DNA segment by infecting lambdaimm434 phages[END_REF][START_REF] Asadulghani | The defective prophage pool of Escherichia coli O157: prophage-prophage interactions potentiate horizontal transfer of virulence determinants[END_REF][START_REF] Ruzin | Molecular genetics of SaPI1-a mobile pathogenicity island in Staphylococcus aureus[END_REF]. We thus verified by PCR that the 3 studied defective prophages do not excise during infection by l. Moreover, as Rac has an active but normally repressed replication origin, we checked by semi-quantitative PCR whether

Author Summary

Temperate bacteriophages (or phages) are bacterial viruses that, unlike virulent phages, have the ability to enter a prophage dormant state upon infection, in which they stably replicate with the bacterial genome. A majority of bacterial genomes contain multiple active or defective prophages, and numerous bacterial phenotypes are modified by these prophages, such as increased virulence. These mobile genetic elements are subject to high levels of genetic exchanges, through which new genes are constantly imported into bacterial genomes. Phageencoded homologous recombination enzymes, or recombinases, are potentially key actors in phage genome shuffling. In this work, we show that gene acquisition in temperate phages is strongly dependent on the presence of sequence homology, but is highly tolerant to divergence. We report that gene exchanges are mainly catalyzed by recombinases found on temperate phages, and show that four different Rad52-like recombinases have a relaxed fidelity in vivo, compared to RecA. This high capacity of exchange speeds up evolution of phages, and indirectly also the evolution of bacteria. its replication was induced upon l infection. We found no evidence of such a phenomenon (Fig. S3).

Recombination between l and the marked defective prophages can result in the integration of cat or kanR antibiotic resistance gene into l (Fig. 1E). As this does not produce a directly detectable phage phenotype, E. coli cells were further lysogenized with the resulting phage, and the proportion of cells lysogenized by WT or antibiotic-resistant recombinant phages was determined by plating on selective antibiotic media (Fig. 1D and Materials and Methods). To be able to detect all lysogenized bacteria, we used a l strain marked with the phleomycine resistance gene ble (Urlble strain, Table S1 and Materials and Methods). This recombination assay enables the estimation of the horizontal gene transfer rate into the l genome when it infects its host.

Mosaicism is produced by homologous recombination l recombinants with Rac and Qin were observed at frequencies around 2610 26 , and were a hundred fold more numerous with Dlp12 (Fig. 1E). Interestingly, the frequencies of recombinants were similar for Rac and Qin despite different extent of identity on comparable lengths (Fig. 1 andS1). In contrast, the recombinant frequency with the control chromosomal gene ilvD, presenting no homology with l genome, was below our detection threshold of 5610 29 . PCR analysis on 20 l recombinants within each of the 3 loci showed that all had incorporated the resistance gene at the expected position for an homologous exchange. 12 to 19 were sequenced to confirm that homologous recombination occurred in the targeted region and to identify the exact junctions (Fig. 2). Among recombinants with Rac, the majority resulted from recombination events within the adjacent 88% identity regions, but one third had recombined on the right in the nearby 95% homology region in tfa.

HR with Rac and Qin leads to the inactivation of l side tail fibers genes (stf and tfa), which reportedly improves phage growth compared to the parent in a soft-agar overlay [START_REF] Gallet | High adsorption rate is detrimental to bacteriophage fitness in a biofilm-like environment[END_REF]. To evaluate if this increases the recombinant frequency by favoring their growth, we performed the same recombination assay with the sequenced lPaPa strain of l, mutated in its stf gene [START_REF] Hendrix | Bacteriophage lambda PaPa: not the mother of all lambda phages[END_REF]. The frequency of lPaPa recombinants with Rac was not significantly different from that with Url (p = 0.18, Student T-test), ruling out the possibility that the advantage of side tail fiber inactivation distorts our conclusions. This can be explained by the low recombination rate: most recombinants statistically arise only during the last (and third) cycle of phage growth, as phage engaging in the third lytic cycle are 10,000 more numerous than those involved in the first one. Sequencing of 12 l-Rac recombinants showed that they all correspond to different hybrids (Fig. 2A), confirming the absence of amplification and also indicating the absence of a recombination hotspot.

The high recombination frequency with Dlp12 is expected for two reasons. First, Dlp12 shares the largest region of homology with l (3353 bp), and secondly, the region of homology contains the l cos site (Fig. 1A). Double-strand DNA breaks at cos sites, created for genome encapsidation, stimulate recombination on its left side, the right side bound by the terminase being protected [START_REF] Thaler | Double-chain-cut sites are recombination hotspots in the Red pathway of phage lambda[END_REF]. Double-strand breaks in both l and Dlp12 might thus stimulate the recombination in this region. Sequencing of 19 l-Dlp12 recombinants indeed revealed a high proportion of junctions in the immediate proximity to cos (10 out of 19 clones, Fig. 2B). 3 out of 19 junctions were nevertheless found on the right side of cos, indicating that double-strand breaks at cos improve but are not necessary for recombination events. Interestingly, all tested l-Dlp12 recombinant phages were active, including those that had replaced the essential l genes R and Rz (lysis) or nu1 (terminase) by those of Dlp12, indicating that these Dlp12 genes are functional in l.

Recombination genes involved in mosaicism

We have shown that genetic exchanges with l are driven by the presence of homologous regions. We next questioned the respective roles on these exchanges of the bacterial and phage HR genes, recA and redb, and also of two other l recombination genes, orf and rap. As redb and reda l mutants have a reduced burst size (Fig. S2), conditions of phage growth on the marked strains were adapted to ensure that the same number of phage generations was realized (see Materials and Methods).

The frequency of recombination with the large, quasi-identical segment of Dlp12 (3.3 kb, 98% identity) was not affected by a single recA deletion, and only 3-fold by a redb deletion (Fig. 3A, blue bars). However, when both recombinase genes were deleted, exchanges were completely abolished. This shows that both RecA and Redb produce recombinants independently of one another, which is indicative of redundant pathways. Within Qin and especially Rac regions, showing more divergence with l, single recA deletion had similarly no effect on recombination frequencies. The single redb deletion however had a pronounced effect on the recombination frequencies (6-and 13-fold respectively, Fig. 3A, orange and green bars), indicating that most recombination events are formed by Redb when homology is reduced, reflecting a lowest activity of RecA on these substrates. The residual recombinants formed in the absence of Redb almost completely disappeared with the double deletion of recA and redb. Only 2 recombinant clones were obtained in the recA redb double mutant, with Rac, and were found by PCR to result from homologous recombination. They might have originated from the expression of the recET recombination genes of Rac. Normally the recET genes are completely repressed in E. coli MG1655, even upon l infection [START_REF] Liu | High-resolution view of bacteriophage lambda gene expression by ribosome profiling[END_REF], and thus cannot promote recombination, but rare sbcA mutations [START_REF] Kolodner | Homologous pairing proteins encoded by the Escherichia coli recE and recT genes[END_REF] or incorporation in l (the so-called l-rev genotype, [START_REF] Mills | Movers and shakers: influence of bacteriophages in shaping the mammalian gut microbiota[END_REF]) can activate them.

Role of other phage encoded recombination promoting genes

The activities of recombinases are stimulated by numerous host or phage-encoded cofactors, which either prepare the substrate for recombination or act at latter stages to resolve the DNA heteroduplex structure. The main partner of Redb is the double-strand-specific exonuclease Reda that transforms doublestrand DNA into single-strand DNA, the substrate for Redb. Reda is dispensable for Redb recombination if the DNA substrate is initially in a single-strand form [START_REF] Ellis | High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides[END_REF].. Here, deletion of reda gene had the same effect as the redb deletion, and basically disabled Red-mediated recombination in Dlp12 and Rac regions (Fig. 3B&C). This finding reveals a need for single-strand DNA formation for Red-mediated recombination in our assay.

We then tested the effects of other recombinase helper proteins on recombinant frequencies. l encodes two such proteins, Orf and Rap, which belong to protein families highly prevalent in phage genomes (respectively 55 and 180 homologues in 465 phage genomes, our unpublished work). Orf and Rap participate in RecA-dependent recombination by supplying a function equivalent to bacterial encoded recombination cofactors, respectively RecFOR and Ruv resolvase [START_REF] Poteete | Modulation of DNA repair and recombination by the bacteriophage lambda Orf function in Escherichia coli K-12[END_REF][START_REF] Poteete | Recombination-promoting activity of the bacteriophage lambda Rap protein in Escherichia coli K-12[END_REF]. Here we asked whether they could also participate in Red recombination, and whether their role was more pronounced on diverged sequences than on easy to recombine long sequences. Single deletion of orf or rap resulted in a not significant 3-fold reduction of l recombination with Dlp12 (Fig. 3B, first set of data). However, in a recA strain, in which all gene exchanges are Red-mediated, recombination was reduced by 10-fold in the Dorf mutant, and by significant 3-fold in the Drap mutant. This result reveals that Orf and to some extent Rap participate in Red-mediated recombination. Finally, in the genetic context where RecA mediates all gene exchanges, the orf deletion had no effect, while the rap deletion decreased recombinant frequency by 300-fold, as expected from an earlier report [START_REF] Hollifield | Efficient RecABC-dependent, homologous recombination between coliphage lambda and plasmids requires a phage ninR region gene[END_REF]. The same genetic analysis in the Rac region assay revealed the same dependencies (Fig. 3C). In conclusion, Orf and to a lesser extent Rap facilitate homologous recombination with l when Redb is the only recombinase, with no indication of an increased role when DNA sequences are more divergent.

Homologous recombination-dependent gene shuffling also occurs with phage W80

In order to determine the generality of our observations with l, we performed similar recombination assays with the lambdoid phage W80. Its genome homology with l is mostly clustered in the capsid region, while the rest of the genome is highly divergent with only few segments of homology [START_REF] Rotman | Genome of Enterobacteriophage Lula/phi80 and insights into its ability to spread in the laboratory environment[END_REF]. W80 encodes a putative recombinase from the Rad52 family, hereafter named RecT W80 , that shares 32% identity at the amino acid level with Redb.

As previously described with l, we detected by Blast search 9 regions of homology between W80 and E. coli MG1655 genomes, and selected 3 of them for the recombination assay: i) the 39 part of the bacterial core gene yecD, of unknown function, presenting 96% mean identity over 300 bp, ii) in the nohD region of the defective prophage Dlp12, sharing 79% identity over 980 bp with the terminase genes nu1 and A, and iii) within the nin region of Dlp12, sharing 67% identity over 500 bp (Fig. 4 A,B,C). For each region, the longest stretch of perfect identity is 116, 41 and 11 bp for yecD, nohD and nin, respectively (Fig. S1B). Homology regions were labeled with the cat gene, and W80 genome was marked with the ble gene. W80 phage was then propagated on the modified E. coli strains and the number of recombinant phages was scored (Fig. 4D).

Recombinants were produced at a frequency of 6610 27 per phage with the yecD locus and 8610 27 with the nohD locus of Dlp12 (Fig. 4D). In the last case, recombination disrupts the essential terminase genes nu1 and A, but this does not prevent encapsidation of recombinant W80 genomes, due to the terminases encoded by the other copies of W80. However, the recombinants formed will not give lytic progeny upon infection of a new host, ensuring that the recombinants counted in the assay are exclusively those formed during the last lytic cycle. As discussed above, the presence of the cos site within the homology region probably increases the production of recombinants. Within the last region, sharing only 67% identity over 500 bp with l, the recombination frequency was below 1610 28 , our detection threshold (ybcN locus, Fig. 3C). PCR analysis revealed that the 12 recombinants scored had incorporated the resistance gene at the locus expected for homologous exchange.

As with l, we then determined the respective role of homologous recombination enzymes RecA and RecT W80 in these genetic exchanges. recA deletion slightly diminishes recombinant frequency by 2-fold on both loci, whereas recT W80 deletion has a pronounced effect on the more diverged sequences (Fig. 3C).

These results show that homology-dependent mosaic formation driven by phage encoded recombinases is not restricted to l and may be a general event among temperate phages encoding their own homologous recombination functions. Exchanges occurred even when the resulting recombinant phages were no longer viable as a lytic phage, and appeared to be only limited by the degree and length of sequence homology.

Like Redb and RecT W80 , Erf D3 and RecT Rac recombinases are efficient on diverged sequences

The capacities of Rad52-like phage encoded recombinases and cofactors were further explored with an intra-bacteriophage recombination assay, which enables the comparison of recombinase activities on the same DNA substrate. We monitored homologous recombination within the l genome, between two inverted 800 bp sequences introduced on each side of the P L promoter (described in Fig. 5A and [START_REF] Martinsohn | The lambda red proteins promote efficient recombination between diverged sequences: implications for bacteriophage genome mosaicism[END_REF]). Briefly, inversion of the P L promoter leads to a detectable phenotypic switch, as it prevents transcription of the red and gam genes, enabling growth on a P2lysogen, contrary to the non-inverted phage. Vice versa, inverted P L Red 2 Gam 2 phage cannot grow on a recA strain, in contrast to the non-inverted wild-type phage. Two different recombination cassettes were used, the inverted sequences being either strictly identical, or 78% identical. The switch was measured in the two directions, and in all cases, the recombination assay was performed by growing phages on a restrictive host for the multiplication of the recombinants, ensuring that the recombinants are produced only during the last generation.

In this study, l redb and reda genes were replaced by other pairs of recombinase genes and their respective associated exonuclease. The first pair is recT and recE (fragment coding for the Cter part of the protein), from the defective prophage Rac. The second pair is the predicted Erf family recombinase gene erf, with its associated predicted exonuclease gene exo, from D3 phage infecting Pseudomonas aeruginosa [START_REF] Iyer | Classification and evolutionary history of the single-strand annealing proteins, RecT, Redbeta, ERF and RAD52[END_REF]. Resulting phages were grown to confluence on a bacterial lawn, and the frequency of recombinants was measured by differential platings. On both the 100% and 78% identical substrates, efficiency of recombination mediated by the RecT/RecE Rac and Erf/Exo D3 pairs was similar to that mediated by l Red proteins (Fig. 5B). First, this demonstrates that the D3 erf gene product is indeed a recombinase, and as efficient as Redb and RecT Rac . For all 3 recombinase/exonuclease pairs, recombination between identical or diverged sequences was respectively 20-and 100-fold higher than in the RecA pathway.

This result extends our previous observation with l and W80, and suggests that during the lytic cycle, the high efficiency and low fidelity of phage-dependent recombination might be a general phenomenon that promotes horizontal gene transfer into phage genomes.

Genomic analysis reveals abundant mosaics among and between temperate and defective phage genomes, but none between virulent and temperate genomes

We thus attempted to quantify the traces that such exchanges might have left on phage genomes on an evolutionary time scale by analyzing a collection of E. coli phages (Table S4). In particular, we examined whether traces of HR events could be detected at the boundaries of recently exchanged DNA fragments. In a previous study of mosaics formed between temperate lambdoid phages [START_REF] Martinsohn | The lambda red proteins promote efficient recombination between diverged sequences: implications for bacteriophage genome mosaicism[END_REF], we showed that half of the mosaics, defined by Blast hits with more than 90% identity, were flanked by at least one region sufficiently similar to be an indication of a region of homology preexistent to the recent exchange identified by the Blast hit. These above background homology regions were hereafter named ''HR traces''. This study was extended and refined here, so as to include more genomes from temperate (24 genomes), defective (34 genomes) or virulent (26 genomes) E. coli phages.

Results are summarized in Table 1. We detected a large number of recently exchanged genomic segments among temperate and defective phages (between 0.8 and 2.4 mosaics per genome pair). No mosaic was found between virulent and temperate or defective phages. We also found a few mosaics between virulent phages (0.06 per genome pair, Table 1). Interestingly, among temperate and defective phages, the density of mosaics, i.e. the numbers of mosaics per 10 kb, was similar (Fig. 6, lower part of the diagonal), suggesting that exchanges do occur at similar frequencies among defective and functional temperate phages. Among temperate phages, two sub-groups not sharing mosaics were found: the larger one contained lambdoid phages (i.e. l-like, e15-like and P22-like), the smaller group contained P2-like, P1 and Mu-like phages. Among pairs involving defective phages, exchanges between these two groups are found, probably because non functional gene combinations are not counterselected in defective prophages. Interestingly, the size distribution of mosaics indicates that small gene fragments are much more often exchanged than complete functional modules (Fig. S5).

We next investigated whether the mosaics had HR traces, i.e. flanking regions that have a lower mean identity than the mosaic but a higher than expected identity (see Figure 7 and Text S1 for the principle of analysis). Briefly, HR traces were detected by complete realignment of the mosaic flanking regions, and identification of above-background levels of nucleotide identity. We found regions suggestive of HR for 32% of the mosaics occurring among temperate phages, 35% of those formed between defective and temperate phages, and 31% for the inter-defective mosaics (Fig. 6, upper side of the diagonal, and Table 1). In most cases, only one, rather than two HR traces were found flanking the mosaics. This may be explained by other mechanisms than HR to generate exchanges but also by successive rounds of exchanges, involving different phage pairs, as illustrated in Fig. S7.

Discussion

It has been known for a long time that related phages can form recombinant hybrids when infecting simultaneously the same bacterial cell, or by recombination with integrated prophages [START_REF] Baker | Recombination and modular exchange in the genesis of new lambdoid phages[END_REF][START_REF] Campbell | Comparative molecular biology of lambdoid phages[END_REF][START_REF] Bouchard | Homologous recombination between a lactococcal bacteriophage and the chromosome of its host strain[END_REF][START_REF] Durmaz | Genetic analysis of chromosomal regions of Lactococcus lactis acquired by recombinant lytic phages[END_REF]. l defective mutants in particular can be rescued by genes present on prophages [START_REF] Hayes | NinR-and red-mediated phageprophage marker rescue recombination in Escherichia coli: recovery of a nonhomologous immlambda DNA segment by infecting lambdaimm434 phages[END_REF]. Here we quantified for the first time these exchanges between phage genomes, and helped to precise the rules that dictate phage mosaicism, demonstrating that they are dependent on homologous recombination enzymes, with a preponderant role for the phage-encoded recombinases when sequences are diverged. Our results suggest that illegitimate recombination is probably much less frequent during phage genome evolution. This high level of exchanges among phage sequences is to be contrasted with the barrier observed against similar exchanges during bacterial recombination: on E. coli plasmids, recombination involving 4% diverged sequences is reduced by 10,000-fold compared to identical sequences [START_REF] Hall | Homologous pairing and strand exchange promoted by the Escherichia coli RecT protein[END_REF], whereas during the lytic cycle, recombination between 12% diverged sequences is reduced only by a 100-fold compared to identical sequences (Fig. 5B).

Mosaic formation is driven by Rad52-like phage recombinases

We demonstrate that phage encoded Rad52-like recombinases play a primordial role in recombining regions sharing only short stretches of homology. Interestingly, on these substrates, all four Rad52-like recombinases that we tested (Redb, RecT Rac , Erf D3 and RecT W80 ) had a higher activity than RecA during l replication. These results strongly support the view that phage Rad52-like recombinases, predominant among E. coli lambdoid phages [START_REF] Bobay | Manipulating or superseding host recombination functions: a dilemma that shapes phage evolvability[END_REF], play a crucial role in genomic shuffling. Interestingly, a recent bioinformatic study showed that the level of mosaicism is higher for phages encoding a recombinase than for others [START_REF] Bobay | Manipulating or superseding host recombination functions: a dilemma that shapes phage evolvability[END_REF], supporting this hypothesis. Whether the same holds true for the two other large families of recombinases encountered in phages (Gp2.5 and Rad51, [START_REF] Lopes | Detection of novel recombinases in bacteriophage genomes unveils Rad52, Rad51 and Gp2.5 remote homologs[END_REF]) remains to be investigated experimentally. Staphylococcus aureus temperate phages exhibit a large level of mosaicism [START_REF] Martinsohn | The lambda red proteins promote efficient recombination between diverged sequences: implications for bacteriophage genome mosaicism[END_REF], and encode indifferently all three types of recombinases, which suggests that the two other families might also have the same property of relaxed fidelity. Further work aiming at comparing these recombinases side by side will allow addressing this point in detail.

The high efficiencies of Rad52-like recombinases on diverged sequences could be due to their higher concentration or activity, compared to RecA, during phage infection. However, on highly homologous sequences, RecA or phage recombination pathways result in a similar yield of recombinants (Fig. 3A and4D). The 10-20 fold higher efficiency of Redb or RecT W80 compared to RecA pathway is unveiled only on more divergent sequences. It could result from activity on shorter perfect sequence identity segments to recombine as compared to RecA. The Minimal Efficient Pairing Segment (MEPS), the minimal size of exact pairing required for efficient exchange in vivo, has indeed been found to be 31-34 bp for RecA [START_REF] Shen | Homologous recombination in Escherichia coli: dependence on substrate length and homology[END_REF] and only 23-27 for Redb [START_REF] Shen | Homologous recombination in Escherichia coli: dependence on substrate length and homology[END_REF][START_REF] Sawitzke | Probing cellular processes with oligo-mediated recombination and using the knowledge gained to optimize recombineering[END_REF]. Our in vivo assays involve sequences that do not have regularly spaced mismatches, so that slight changes in the number of segments above MEPS size may have drastic effects. Interestingly, Li and collaborators have recently reported that recombineering with regularly spaced, diverged oligonucleotides is effective up to 1 mismatch every 5 nucleotide, which corresponds to a mean divergence of 17% [START_REF] Li | Bacterial DNA polymerases participate in oligonucleotide recombination[END_REF], and fits nicely our observations. The low sensibility to divergence of phage recombinases could also result from different sensitivities to methyl-directed mismatch repair (MMR), comprising MutSLH proteins. Indeed, the high fidelity of homologous recombination is caused not only by the intrinsic properties of the enzymes, but also by MMR inhibition of exchange if mismatches are present in recombination intermediates [START_REF] Radman | DNA mismatch repair systems: mechanisms and applications in biotechnology[END_REF]. Classically, defects in MMR provoke a 100-fold increase of HR, either in RecA [START_REF] Elez | The frequency and structure of recombinant products is determined by the cellular level of MutL[END_REF] or Redb pathways [START_REF] Costantino | Enhanced levels of lambda Red-mediated recombinants in mismatch repair mutants[END_REF]. However HR is less affected by the MMR during the l lytic cycle: RecAdependent recombination is enhanced by only 2 to 8 fold in a mutS background, depending on the level of divergence, and Redb-dependent recombination is not affected at all [START_REF] Martinsohn | The lambda red proteins promote efficient recombination between diverged sequences: implications for bacteriophage genome mosaicism[END_REF]. Whether mismatch repair is titrated or inhibited during the phage lytic cycle is an open question that deserves further inquiry.

In l, red mutants have a 5-fold reduced burst size (Fig. S2), which at present remains unexplained (see [START_REF] Poteete | What makes the bacteriophage lambda Red system useful for genetic engineering: molecular mechanism and biological function[END_REF] for a review discussing this point). Our observation that RecA substitutes completely for Red for HR on identical sequences seems to exclude that the loss of viability of red mutants is due to a strict HR defect. RecBCD pathway is inactive in l infected cells as l lacks Chi sites and moreover express a RecBCD inhibitor. Further, we could not detect any synergy of the red and recA mutations on plaque sizes (Fig. S4). Whether Red impacts replication or any other stage of the phage cycle remains to be investigated, as well as why W80 recombinase does not impact W80 plaque size.

Biochemical evidence suggests that Sak and Sak3, two Rad52like recombinases of lactococcal phages, act as cofactors of RecA [START_REF] Ploquin | Functional and structural basis for a bacteriophage homolog of human RAD52[END_REF][START_REF] Sokol | The intestinal microbiota in inflammatory bowel diseases: time to connect with the host[END_REF]. Some in vivo work also pointed to such a role for Redb on non-replicating l genomes [START_REF] Sokol | Crypt abscess-associated microbiota in inflammatory bowel disease and acute self-limited colitis[END_REF]. In the present work, the phage and host recombinases appear rather to work independently from each other, and redundantly on highly similar sequences.

The RecA cofactor function of phage-encoded Rad52like proteins may therefore be a minor activity in vivo. This situation is contrasted with the yeast-encoded Rad52 protein, which is essentially known for its Rad51 cofactor activity. However, Rad52 also performs some repair reactions in a Rad51 independent way [START_REF] Mott | RAD51-independent inverted-repeat recombination by a strand-annealing mechanism[END_REF]. Whether these Rad52 activities are tolerant to diverged DNA is unknown at present. Interestingly, another kind of mobile genetic elements that present genomic mosaicism, the integrative conjugative elements of the SXT/ R391 family, has been reported to encode a recombinase of the Rad52 family (named s065) [START_REF] Wrzosek | Bacteroides thetaiotaomicron and Faecalibacterium prausnitzii influence the production of mucus glycans and the development of goblet cells in the colonic epithelium of a gnotobiotic model rodent[END_REF]. It was found to act in the formation of hybrid ICEs, independently of RecA. The assay involved 95-97% identical sequences, and RecA was the dominant pathway. Whether s065 becomes dominant when recombination involves more diverged sequences, remains to be investigated. A: Principle of the assay: two 800 bp homologous sequences (oxa genes, as in [START_REF] Martinsohn | The lambda red proteins promote efficient recombination between diverged sequences: implications for bacteriophage genome mosaicism[END_REF]), inversely repeated, represented by the red and yellow rectangles, are inserted so as to flank the P L promoter in the l genome. Inverted sequences are either 100% or 78% identical. When homologous recombination occurs between the repeated sequences, the P L promoter is inverted, which leads to a phenotypic switch, because the red and gam genes are no longer expressed (see Materials and Methods). B: Recombination frequencies scored with three different pairs of phage recombinase/exonuclease, and compared to RecA pathway. The recET (from Rac prophage) and erf/ exo (from phage PA73) genes were substituted in place of the l red genes. Values shown for redab and recA pathways are those reported in [START_REF] Martinsohn | The lambda red proteins promote efficient recombination between diverged sequences: implications for bacteriophage genome mosaicism[END_REF]. Mean 6 standard deviation of at least 3 independent recombination assays are indicated. doi:10.1371/journal.pgen.1004181.g005

Contribution of accessory proteins Orf and Rap to recombination

Interestingly, both Orf and Rap proteins have numerous homologs among temperate phages, and the presence of Orf is strongly associated with the presence of a recombinase. Notably, W80 possesses homologs of both orf (gp53) and rap. Rap (recombination adept with plasmid) increases RecA-dependent recombination between l and a plasmid sharing perfect identity by a 100-fold [START_REF] Hollifield | Efficient RecABC-dependent, homologous recombination between coliphage lambda and plasmids requires a phage ninR region gene[END_REF]. In line with this result, we found that in the RecA pathway, rap deletion results in a 500-fold decrease in recombination with Dlp12. With the Rac substrate, where the activity of the RecA pathway is minor, rap deletion results in a further 10-fold decrease, again underlining the importance of rap in this pathway. For Redb-dependent recombination, the decreases due to rap deletion were only 3 and 6-fold for Dlp12 and Rac substrates, respectively. This is probably related to the fact that the Rap substrates, Holliday junctions, are not necessarily formed by Redb Indeed, Redb principally recombines by a strand assimilation mechanism, which consists in the single-strand annealing between Redb-bound single-strand DNA and the lagging strand template of a replication fork, a reaction that does not need a resolution step [START_REF] Court | Genetic engineering using homologous recombination[END_REF][START_REF] Poteete | Involvement of DNA replication in phage lambda Redmediated homologous recombination[END_REF][START_REF] Maresca | Single-stranded heteroduplex intermediates in lambda Red homologous recombination[END_REF][START_REF] Mosberg | Lambda red recombineering in Escherichia coli occurs through a fully single-stranded intermediate[END_REF]. On the contrary, RecA catalyses mainly strand invasion reactions, which generate Holliday junctions [START_REF] Noirot | DNA strand invasion promoted by Escherichia coli RecT protein[END_REF][START_REF] Rybalchenko | Strand invasion promoted by recombination protein beta of coliphage lambda[END_REF]. The Rap protein acts essentially independently from Redb, a conclusion which agrees nicely with the independent genomic distribution of these two genes.

Orf is involved in displacing of SSB from single-stranded DNA, which facilitates RecA binding. We found that orf deletion decreases Redb dependent recombination by 10-fold, and does not affect the RecA pathway. This is in line with the strong association observed between orf and recombinase genes in phage genomes. This Orf effect might reflect the proportion of cases where Redb enters in competition with SSB. As Redb loads onto single-strand DNA immediately after Reda, the two proteins being supposedly in interaction, the Orf activity to remove SSB should not be essential, as SSB might not be present on most of the substrates recombined by Redb.

Quantification of mosaics and traces of homologous recombination in phage genomes

The presence of all these recombination genes in phage genomes influences their long term evolution. Indeed, here we add evidences that HR leaves frequent traces among phage genomes. We found recently formed mosaics -defined as sequences sharing more than 90% identity -between temperate and defective genomes, but also a few ones among virulent phages. Interestingly, recent gene exchanges were identified only inside the lambda-like group or the P1-P2-Mu group, but not between each group. Traces of homology flanking the mosaic were detected in ,30% of the mosaics between temperate phages and/or defective prophages. Whether the remaining 70% mosaics were formed by HR but have shorter or masked traces (Fig. S7), or were created by non-homologous recombination remains an open question.

Remarkably, no recent mosaics were found between temperate and virulent phages infecting E. coli. In fact, a well documented case of ancient horizontal gene transfer among a large group of unrelated temperate and virulent phages exists, and concerns the side tail fiber (stf) genes [START_REF] Montag | A component of the side tail fiber of Escherichia coli bacteriophage lambda can functionally replace the receptorrecognizing part of a long tail fiber protein of the unrelated bacteriophage T4[END_REF][START_REF] Haggard-Ljungquist | DNA sequences of the tail fiber genes of bacteriophage P2: evidence for horizontal transfer of tail fiber genes among unrelated bacteriophages[END_REF][START_REF] Sandmeier | DNA inversion regions Min of plasmid p15B and Cin of bacteriophage P1: evolution of bacteriophage tail fiber genes[END_REF]. However, this case is very specific, as these genetic exchanges are most probably driven by stf associated phage invertases that catalyze site-specific recombination [START_REF] Sandmeier | Acquisition and rearrangement of sequence motifs in the evolution of bacteriophage tail fibres[END_REF]. The very low potentiality of virulent phages to acquire genes from temperate phages, which often carry virulence genes, is reassuring for the development of phage therapy as a means to combat bacterial infections. It should be noted however that some virulent phages are in fact former temperate phages that lost their lysogeny module. The case is well documented among dairy phages [START_REF] Chopin | Analysis of six prophages in Lactococcus lactis IL1403: different genetic structure of temperate and virulent phage populations[END_REF]. Such ''virulent'' phages, that should rather be named ''ex-temperate'', do exchange sequences with temperate and defective prophages [START_REF] Chopin | Analysis of six prophages in Lactococcus lactis IL1403: different genetic structure of temperate and virulent phage populations[END_REF] and should therefore be avoided for phage therapy, as already indicated [START_REF] Miquel | Faecalibacterium prausnitzii and human intestinal health[END_REF]. To ascertain the choice of virulent phages for therapeutic use in the future, it may be relevant to conduct an analysis similar to the one presented here, by looking for mosaics (i.e. Blast matches above 90% identity) between the selected (and sequenced) phage, and all possible sequenced phages and prophages infecting the targeted bacterial species.

Consequences for evolution

The relaxed exchange of genetic information among phages might result from different selective pressures acting on viruses as compared to bacteria. The level of HR is indeed the result of contradictory demands. Recombination between homologous sequences must be an efficient process to repair rapidly DNA lesions. On the other hand, recombination between different homologous loci, by creating genomic rearrangement, can destroy chromosomal integrity and functional gene associations and must be avoided. This problem is especially pronounced in eukaryotes, which contain repeated sequences with slight polymorphism, that are prone to recombine together, but bacterial chromosomes also possess repeated elements and related genes. Phage genomes, however, do not possess repeated sequences usually, and can thus better tolerate recombination between diverged sequences without risking chromosomal rearrangements. This might explain the phage tolerance to low fidelity recombination, which then accidentally contribute to rapid phage evolution. Alternatively, as recombination of diverged sequences is sometime advantageous for phages, it is tempting to speculate that it is under positive selection. First, high levels of shuffling inside genes can generate new functions (reviewed in [START_REF] Long | The origin of new genes: glimpses from the young and old[END_REF]). We found that small sequences of 100 bp within genes are frequently exchanged. This mechanism to generate new genes could explain the much larger viral gene repertoire as compared to bacterial gene repertoire. Secondly, creation of new gene combinations is likely valuable in numerous functions to allow greater phage propagation, e.g., escape from CRISPR-mediated bacterial immunity systems [START_REF] Sorek | CRISPR-mediated Adaptive Immune Systems in Bacteria and Archaea[END_REF] or expansion of bacterial host range [START_REF] Haggard-Ljungquist | DNA sequences of the tail fiber genes of bacteriophage P2: evidence for horizontal transfer of tail fiber genes among unrelated bacteriophages[END_REF][START_REF] Lwoff | Lysogeny[END_REF].

Temperate phage genes are alternatively submitted to very different selective pressures depending on the nature of their replicating cycle. Once integrated, prophage DNA becomes subject to the selective forces working on the bacterial chromosome. This explains that temperate phages constitute a reservoir of genes that improve bacterial phenotypes by lysogenisation [START_REF] Miquel | Faecalibacterium prausnitzii and human intestinal health[END_REF][START_REF] Brussow | Phages and the evolution of bacterial pathogens: from genomic rearrangements to lysogenic conversion[END_REF]. Likewise, defective prophages, that were long considered to be mere genetic junk, are now known to confer a broad range of beneficial phenotypes to the bacterial hosts, with respect to virulence, stress resistance, or even mutation rate [START_REF] Brussow | Phages and the evolution of bacterial pathogens: from genomic rearrangements to lysogenic conversion[END_REF][START_REF] Panis | Insights into the functions of a prophage recombination directionality factor[END_REF][START_REF] Wang | Cryptic prophages help bacteria cope with adverse environments[END_REF][START_REF] Rabinovich | Prophage Excision Activates Listeria Competence Genes that Promote Phagosomal Escape and Virulence[END_REF]. The present study illustrates that inversely, prophage remnants can be a reservoir of functional lytic cycle genes for temperate phages. Dlp12 lysis module, helpful in certain strains of E. coli for biofilm development [START_REF] Kolodner | Homologous pairing proteins encoded by the Escherichia coli recE and recT genes[END_REF], is also active in l for lysis. The constant exchange of genes between prophage remnants entrapped in host chromosome and active phages, via homologous recombination, blurs even more the distinction between evolutionary pressures acting on temperate phages and their bacterial host, tightly links their evolution, and indirectly accelerates bacterial evolution itself.

Materials and Methods

Bacterial and phage strains

All phage and bacterial strains are listed in Table S1. Unless specified, all gene replacements, deletions or insertions were done by recombineering as already described [START_REF] Datsenko | One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products[END_REF], with primers specified in Table S2.

The MG1655 stfR::cat (in Rac prophage) and tfaQ::cat (in Qin prophage) were constructed by inserting the cat cassette from the pKD3 plasmid into the respective genes with the stfR::cat and tfaQ::cat oligonucleotide pairs listed in Table S2, respectively. The MG1655 ybcV::Kan (in Dlp12 prophage) was constructed by P1 transduction from the Keio collection strain ECK0550 [START_REF] Baba | Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection[END_REF].

The Urlble phage was constructed from Url isolated from the E. coli K12 ancestral strain. The phleomycine/bleomycine resistance gene ble, cloned under the Bacillus subtilis promoter psacB, taken from plasmid pUCphleo (gift from E. Dervyn), was introduced between the tfa and ea47 genes of l, and oriented as tfa. The presence of ble does not modify phage growth (Fig. S2), nor the frequency of lysogenization. Complete sequencing of Urlble showed that no important mutation other than the presence of the ble gene and the expected frameshift in the stf gene [START_REF] Hendrix | Bacteriophage lambda PaPa: not the mother of all lambda phages[END_REF] differentiate our strain from the sequenced lPaPa strain (Table S2).

Deletions of redb, reda, orf and rap genes were done by recombineering in E. coli K12 Urlble. In the case of the deletion of redb, a RBS was introduced to maintain the expression of reda, as the two genes are overlapping and reda RBS is inside redb.

Phage W80 was isolated in our laboratory from a strain contamination. Phage W80ble was constructed by introducing the psacB-ble construct after gp63, and in the same orientation.

The lNec9 and 10 were constructed by replacing reda and redb by the Rac-encoded recE and recT genes, in lNec4 and lNec6, respectively (phage strains listed in Table S1). To do this, the fragment corresponding to the C-terminal (from a.a. 588) of RecE and the full recT gene was added into pKD4 at the BmgBI site, to give pJA100. The PCR fragment generated from pJA100, using the pair of oligonucleotides pKD4 (Table S3) was then introduced between the gam and orf60a genes of lNec4 and lNec6 by recombineering [START_REF] Datsenko | One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products[END_REF]. To construct lNec11 and lNec12, reda and redb were replaced by erf and exo genes from D3 (a P. aeruginosa phage) in lNec4 and lNec6, respectively. First, these two genes were cloned into pKD4 (pJA82, constructed with erf/exo oligonucleotides described in Table S3) at the BmgBI site, then the PCR fragment generated from this plasmid (using the oligonucleotides pKD4 described in Table S3) was introduced between the gam and orf60a genes of lNec4 and lNec6, following the same steps as for the recET constructions. Final constructs were verified by sequencing.

Measure of phage growth

Burst size was determined on E. coli MG1655 bacteria growing exponentially in LB broth supplemented with maltose (0.2% w/v) and magnesium (MgSO4 at a concentration of 10 mM). When OD 600 reached 0.2, 10 ml of the culture were concentrated 10 times, and the phage added at a multiplicity of infection of 0.002. The mix was incubated for 7 minutes at 37uC and then diluted 100-fold and 10,000-fold in LB at 37uC. The number of nonadsorbed phage at the start of the phage growth was evaluated by plaque assay after bacterial centrifugation. Samples of the two dilution mixes were taken repeatedly throughout time and assayed immediately for plaque-forming units. The burst size is the factor between final phage number and initial phage number, subtracting the number of non-adsorbed phage at time of dilution.

Plaque size was determined after overnight growth on a layer of E. coli MG1655 bacteria embedded into top agarose supplemented with maltose and magnesium (2 g/L agarose, 10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl, 10 mM MgSO4, 2 g/L maltose). Plates imaging and analysis was realized with the Colony Doc-it imaging station (UVP, Upland, Canada) with the same settings for all plates.

Phage-prophage recombination assay

The phage to be tested was multiplied on the appropriate E. coli strains on plates. Briefly, 10 4 (for Red+ l strains and W80 strains) or 10 6 (for Dredb l strains) PFU were mixed to 100 ml of overnight bacterial culture grown on LB maltose. After 5 minutes of incubation, 4 ml of top agar containing MgSO4 10 mM were added and then poured on a fresh LBA plate containing 0.2% of glucose. After 6 to 8 hours of incubation at 37uC, when lysis was confluent, 5 ml of water were poured on the top of the plates and incubated at 4uC for two hours. The phage supernatant was then recovered and filtrated at 0.2 mm. Stocks were around 10 10 PFU/ ml for Red positive strains and 10 9 PFU/ml for Dredb or Dexo strains. A theoretical calculation based on the measured burst sizes in liquid medium allowed to estimate that under such conditions Redb + and Redb 2 phages performed similarly 2.8 generations during the recombination assay. W80 experiments were performed similarly but with few differences: no maltose was added to the medium, and the same amount of RecT+ and RecT2 phages (10 4 ) were used for inoculation as no difference in plaque sizes was observed between the two genotypes.

Read-out of the phage-prophage recombination assay by lysogenization 1 ml of E. coli MG1655 culture growing in LB+0.2% maltose+ MgSO4 10 mM was added to the phage stock in which recombinants were produced (m.o.i = 1) when the OD 600 of the culture reached 1. The mix was then incubated for 1.5 hours at 37uC in a closed 2 ml tube. The total number of lysogenized bacteria was measured by plating the bacteria at the appropriate dilution on phleomycine (5 mg/L) plates. Recombinant phage concentration was estimated by plating on either chloramphenicol (20 mg/L) or kanamycine (50 ml/L) plates. In these conditions, 5 to 10% of the bacteria were lysogenized, as indicated by the proportion of bacteria that acquired phleomycine resistance. Recombinant frequencies are the ratio of chloramphenicol or kanamycine resistant bacteria on phleomycine resistant bacteria. Figures given are an average of at least three independent recombination assays, followed for each of them by at least 3 read outs by lysogenisation.

Analyses of recombinant clones

PCR amplification of the l genomic region susceptible of containing the resistance gene acquired from the host chromosome, if recombination was guided by homology, were realized on more than 20 colonies per genotype (oligonucleotide sequences are given in Table S3). For each colony tested, a fragment at the expected size was obtained. About 35% of the colonies gave an additional band corresponding to the native size of the l region tested. A PCR using divergent oligonucleotides at int and ea59 (that give a product if l is excised or integrated in multiple copies) allowed confirming that these colonies were polylysogens. As the same proportion of polylysogens was found for each mutant, this phenomenon was neglected for the calculation of the frequency of recombinants.

P L inversion assay on phage l 2610 6 lNec phages (100 mL) were incubated 5 min with 2610 7 (500 mL) of exponentially growing C600 recA cells. Then 5 mL of top agar containing 10 mM MgSO4 was poured onto the mix and plated on LB plates, which were incubated 6 h at 37uC, until confluent lysis. 5 mL of a 10 mM MgSO4 solution were poured on the lysed plates, and the whole top agar layer was recovered, vortexed, and centrifuged for 10 min at 4uC. The supernatant, containing the phage, was then recovered and filtered at 0.2 mm. To estimate the amount of recombinants produced during lytic growth on plates, the stock was titrated on C600 recA strain to count parental phage, and on a C600 P2 lysogen strain, where only recombinant phage can grow, to count the recombinants. The frequency of recombination was then calculated by the number of recombinant PFU divided by the number of PFU corresponding to parental phages, counted on the recA strain.

Bioinformatics analysis of mosaicism

A set of 50 non-redundant genomes (less than 92% overall identity or coverage ,80%) of phages infecting E. coli, and having either temperate (n = 24) or virulent (n = 26) lifestyles were chosen (see Table S4). The 34 non-redundant defective prophages were those of strains MG1655 and 0157:H7 Sakai [START_REF] Asadulghani | The defective prophage pool of Escherichia coli O157: prophage-prophage interactions potentiate horizontal transfer of virulence determinants[END_REF] as well as the prophages identified in IAI39 E. coli strain (all contained Insertion Sequences (IS) in genes important for the phage life cycle, and were therefore classified as defective). The analysis was focused on recent exchanges only (mosaics with more than 90% identity), because it aimed at detecting traces of recombination in mosaic flanking sequences. Among the selected phages, some pairs were too closely related to contribute to the analysis: when more than 50% of the smaller genome of the pair shared more than 70% average nucleotide identity with its partner, the pair was excluded (black squares in Fig. 6, the strategy used to examine phage relatedness is shown Fig. S7). To detect recent exchanges, a Blastn was run on each genome pair, and all hits of a minimal size of 100 bp and sharing more than 90% identity were selected. Among these hits, some corresponded to IS sequences: they contributed for 9% of the total hits found among temperate phages, but as much as 33% among the defective phage pairs. The rest of the hits were named mosaics.

Under the hypothesis of mosaics formed by homologous recombination between diverged sequences, the scenario depicted in Fig. 7 is supposed to take place. Two ancestral phages A and B sharing in average 60% identity except for some more conserved regions at 80% identity, recombine across these 80% identical sequences (light grey). As a consequence, the new piece of DNA in the recombined phage C, when compared to its parent B providing the mosaic, exhibits a 100% identity region (dark grey), flanked by the two 80% identical sequences, above the background level of 60% identity shared by the two genomes at the time of exchange, and somewhat less at the time where genomes are analyzed. Following this scheme, to detect traces of homologous recombination at the vicinity of the mosaics, pairs of 2 kb-long sequences flanking the mosaics were realigned by dynamic programming, and the identity level of successive 50 bp-long windows along the alignment was measured. The 50 bp length was chosen as a close value to the minimum required for Redb recombination (30 bp). The probability to encounter such HR traces at random (last line of Table 1) was calculated as described in Text S1. Figure S4 Plaque size distribution of l mutants in the presence or absence of RecA in the host. To determine the effect of phage mutations on growth, we used the plaque size method. As all phage parameters like virion size, adsorption rate and latency period are equal in other respects, a reduction in burst size should directly translate into a smaller plaque of lysis on a lawn of bacteria. The same phage mutants were thus grown on MG1655 or isogenic recA lawns. redb and reda mutants give a 2.5-fold smaller plaque area, but no difference in plaque size for orf and rap mutants is observed. On a recA lawn, a uniform and modest 15% decrease of plaque size was found for all mutants, probably reflecting the lower growth rate of the recA mutant. Interestingly, on a recA host, the redb deleted phage makes plaques of a similar size compared to the WT host, whereas homologous recombination on phage DNA is abolished. This suggests that RecA, while being essential for HR when a l red phage is used, is dispensable for growth of such l red phage, and therefore that the Red function in growth is distinct from its role in homologous recombination. (DOCX)

Supporting Information

Figure S5 Distribution of mosaic sizes in the 4 categories of comparisons. (DOCX)

Figure S6 Strategy to determine the phage genome pairs to be excluded from the mosaic analysis. Mosaic analysis was focused on recent exchanges (mosaics with more than 90% identity), because it aimed at detecting the relatedness of the mosaic flanking sequences. The genome pairs too closely related to permit detection of such flanking sequences needed to be eliminated. We reasoned that if the average nucleotide identity (ANI) of the genome pair was above 70%, no clear signal above the ANI value, and different from the mosaic signal would be detected. We also specified that at least 50% of the genomes needed to be included for the measurement of the ANI value, to be relevant. To measure the ANI of each genome pair, the method of Goris et al. was adapted [START_REF] Goris | DNA-DNA hybridization values and their relationship to whole-genome sequence similarities[END_REF]. Instead of using standard Blast parameters, with which ANI below 80% are seldom generated, we used the following values: cost to open a gap: 5, cost to extend a gap: 2, penalty for nucleotide mismatch : -4, reward for nucleotide match : 3, word size : 10. This allowed reaching ANI values down to 60%. ANI values as a function of genome coverage for temperate and defective pairs (A), and for virulent pairs (B) are shown. The virulent pairs of genomes are dominated by T4-like phages, which group nicely into discrete sets of ANI values. In order to avoid cutting within a group, virulent pairs with ANI value above 68% and coverage above 48% were removed from the mosaic analysis (red frame). In a marked contrast, temperate/defective pairs form a cloud of points, among which the subset with ANI value above 70% and coverage value above 50% are the pairs removed from the analysis (red frame). ANI analysis was performed for pairs of genomes listed in TableS4 having mosaics. In the black frames of Figure S6, (.92% id, .80% cov), genomes are so similar that they were completely removed from the analysis (indicated with * as redundant in the last column of Table S4). (DOCX)

Figure S7

Scheme for the masking of homologous recombination traces. Genome A/B is the product of a recombination between A and B in regions of homology. As described in Fig. 7, alignment of the two genomes should reveal traces of homologous recombination on both sides of the mosaic. However, recombination of the A/B hybrid with a third genome, C, can mask one of the two HR traces. Alignment of A/B/C hybrid with the ancestral B genome will reveal only one trace of homologous recombination. (DOCX)

68

Text S1: Probability to encounter traces of HR at random around mosaics :

At the vicinity of the mosaics, pairs of 2 kb-long sequences flanking the mosaics were aligned, and the identity level of successive 50 bp-long windows along the alignment was measured. An example of the identity profile flanking a mosaic is shown below. In red, the mosaic, in orange, the flanking region that could be a trace of HR, and in yellow some other region of higher-than-background similarity, that does not flank a mosaic. In blue, the background level of identity of the two regions compared.

Window # in the alignment

From such identity profiles, b, the background level of identity shared by the two genomes (taken from the third median value) is derived. Next, regions having a higher-thanbackground level of identity, namely above b+10%, and placed next to the mosaic are classified as "trace of HR", whereas the other such regions that are distant from the mosaic are classified as "bumps". In the above example, the mosaic flanking region has 1 trace of HR and 4 bumps. For a given category of phages being compared, the total number of mosaics found is Nm, the total number of traces of HR is Nth, and the total number of bumps is Nb.

To estimate whether Nth is significantly above Nr, the number of bumps expected at random just next to the mosaic, the following calculation is made: we model the occurrences of bumps along the concatenated realigned sequences by a Poisson process with intensity i estimated by the number of bumps Nb divided by the cumulative length of all sequences realigned. The number Nr of bumps expected at random in the 100 bp next to all mosaics of the group is then distributed according to a Poisson distribution with mean Nm x 100 x i. It implies that the probability to observe as much as Nth traces of HR is given by the tail of this % identity

+ G IR L-K + 1 17183 A -> G J E 560 -> G 18835 T -> G J Y 1111 -> D 20661 A -> G stf after 20835 + C stf + 1 21714 G -> A stf S 689 -> N 22445 A -> G tfa K 158 -> E 22597-22633 deletion -> psacB-phleoR 25662 T -> G ea59 M 438 -> L 31016 T -> C orf61 34934 A -> G sieB 45618
T -> C R IR: intergenic region *: Absence of change indicates a synonymous mutation. In addition to the expected C addition in stf, that restores the reading frame and therefore intact side tail fibers in Urλ, one deletion and two insertions are observed in intergenic regions (IR). Among the 8 substitutions, 3 are neutral, and 4 of the 5 non-synonymous changes are in tail component genes.

FIGURE S4

+ -+ -+ -+ -+ -+ -+ - WT Δredβ Δredα Δorf Δrap Δredβ Δredβ Δorf Δrap RecA Figure S4
Plaque size distribution of l mutants in the presence or absence of RecA in the host. To determine the effect of phage mutations on growth, we used the plaque size method. As all phage parameters like virion size, adsorption rate and latency period are equal in other respects, a reduction in burst size should directly translate into a smaller plaque of lysis on a lawn of bacteria. The same phage mutants were thus grown on MG1655 or isogenic recA lawns. redb and reda mutants give a 2.5-fold smaller plaque area, but no difference in plaque size for orf and rap mutants is observed. On a recA lawn, a uniform and modest 15% decrease of plaque size was found for all mutants, probably reflecting the lower growth rate of the recA mutant. Interestingly, on a recA host, the redb deleted phage makes plaques of a similar size compared to the WT host, whereas homologous recombina-tion on phage DNA is abolished. This suggests that RecA, while being essential for HR when a l red phage is used, is dispensable for growth of such l red phage, and therefore that the Red function in growth is distinct from its role in homologous recombination. Mosaic analysis was focused on recent exchanges (mosaics with more than 90% identity), because it aimed at detecting the relatedness of the mosaic flanking sequences. The genome pairs too closely related to permit detection of such flanking sequences needed to be eliminated. We reasoned that if the average nucleotide identity (ANI) of the genome pair was above 70%, no clear signal above the ANI value, and different from the mosaic signal would be detected. We also specified that at least 50% of the genomes needed to be included for the measurement of the ANI value, to be relevant. To measure the ANI of each genome pair, the method of Goris et al. was adapted [86]. Instead of using standard Blast parameters, with which ANI below 80% are seldom generated, we used the following values: cost to open a gap: 5, cost to extend a gap: 2, penalty for nucleotide mismatch : -4, reward for nucleotide match : 3, word size : 10. This allowed reaching ANI values down to 60%. ANI values as a function of genome coverage for temperate and defective pairs (A), and for virulent pairs (B) are shown. The virulent pairs of genomes are dominated by T4-like phages, which group nicely into discrete sets of ANI values. In order to avoid cutting within a group, virulent pairs with ANI value above 68% and coverage above 48% were removed from the mosaic analysis (red frame). In a marked contrast, temperate/defective pairs form a cloud of points, among which the subset with ANI value above 70% and coverage value above 50% are the pairs removed from the analysis (red frame). ANI analysis was performed for pairs of genomes listed in TableS4 having mosaics. In the black frames of Figure S6, (.92% id,.80% cov), genomes are so similar that they were completely removed from the analysis (indicated with * as redundant in the last column of Table S4).

Figure S7 Scheme for the masking of homologous recombination traces.

Genome A/B is the product of a recombination between A and B in regions of homology. As described in Fig. 7, alignment of the two genomes should reveal traces of homologous recombination on both sides of the mosaic. However, recombination of the A/B hybrid with a third genome, C, can mask one of the twoHR traces. Alignment of A/B/Chybrid with the ancestral B genome will reveal only one trace of homologous recombination.

INTRODUCTION

Homologous recombination is a key mechanism to the survival of all living organisms and a major force to their evolution. The recombinase is key to the process and conserved in all the three living kingdoms. In Bacteria, this recombinase is named RecA, and its mechanism of action is well described, particularly in Escherichia coli (see (Kuzminov, 1999;[START_REF] Cox | Motoring along with the bacterial RecA protein[END_REF] for reviews). In Eukaryota, its homologue Rad51 is also well studied in many organisms, from yeast to humans (Krogh and Symington, 2004;[START_REF] Krejci | Homologous recombination and its regulation[END_REF]. In Archaea, the RadA protein shares homology with RecA and Rad51 (in the 30% identity range) and shares the same activity (Seitz et al., 1998;[START_REF] Haldenby | RecA family proteins in archaea: RadA and its cousins[END_REF]. Briefly, these proteins bind ssDNA, form a filament on it and invade dsDNA to search homology and anneal the two DNA, by a so-called strand invasion mechanism. Recently, for RecA and Rad51, these activities were dissected using single molecule assays [START_REF] Van Der Heijden | Real-time assembly and disassembly of human RAD51 filaments on individual DNA molecules[END_REF]van der Heijden et al., 2008;[START_REF] Forget | Single-molecule imaging brings Rad51 nucleoprotein filaments into focus[END_REF]2012).

Redβ of the phage λ, infecting E. coli, promotes homologous recombination by a mechanism distinct from RecA, namely single strand annealing (SSA). In vitro, it binds to ssDNA and facilitates annealing of two complementary ssDNA ( (Kuzminov, 1999) for review). It is proposed to act in vivo by first loading on an ssDNA fragment (prepared by its cognate exonuclease Redα), and then promoting the annealing of this ssDNA fragment onto the lagging strand matrix at the replication fork, taking the place of an Okazaki fragment (Maresca et al., 2010). Furthermore, Redβ also promotes some level of strand invasion in vitro, under conditions of low of magnesium and AT-rich DNA template (Komori et al., 2000). Some in vivo studies are also suggesting that Redβ and the related RecT recombinase perform strand invasion, albeit less efficiently than SSA (Muyrers et al., 2000;Martinsohn et al., 2008).The efficiency of Redβ in vivo is high, and we showed recently that homologous recombination was as efficient for Redβ and RecT as for RecA on perfectly identical sequences, and even 10 to 100 fold more efficient compared to RecA, on diverged sequences (up to 22% divergence) (Martinsohn et al., 2008;[START_REF] De Paepe | Temperate phages acquire DNA from defective prophages by relaxed homologous recombination: the role of Rad52-like recombinases[END_REF]. This suggests that phage-encoded recombinases of this family have a relaxed fidelity, which may be at the root of the remarkable mosaicism of some temperate phage genomes.

Redβ was recently recognized to share distant similarity with the N-terminal domain of the eukaryotic Rad52 protein, together with other phage recombinases of the Erf and Sak families (Ploquin et al., 2008;Erler et al., 2009;Lopes et al., 2010). Altogether these three families of Rad52like proteins constitute the most populated superfamily of phage recombinases. Whereas the main activity of Rad52 in S. cerevisiae is that of a cofactor of Rad51, it should be noted that in phages, the protein is fully active as a recombinase by itself in vivo: it acts equally well in wild type strains and in composed by 3 full-size paralogs of Rad51 (Rad51B, C and D) and a core-only Rad51 (Xrcc2) can promote SSA in human (Yokoyama et al., 2004).

Here, we report a genetic characterization of sak4 encoded by phage HK620 (infecting E. coli) and a biochemical characterization of Sak4 of phage PA73 of P. aeruginosa. We show that a distant homolog of SSB is present next to the sak4 gene in most phages, and using a ssDNA recombineering assay, we found that Sak4 HK620 promotes SSA in vivo, provided its cognate SSB is co-expressed with it.

Similarly to Redβ λ , Sak4 HK620 is active on diverged DNA substrates, up to 12% in vivo. In vitro, Sak4 proteins turned out to be insoluble, except for the GST-Sak4 PA73 fusion, which was further studied. It was unable to perform the typical, RecA-dependent strand exchange reaction, contrary to RecA, but performed efficient SSA, pairing up to 20% diverged, 81nt long oligonucleotides in vitro. Its cognate SSB had affinity to ssDNA, but did not enhance any of its biochemical properties.

MATERIAL AND METHODS

Strains & oligonucleotides and proteins

Strains of E. coli used in this study are described in Table 1. Oligonucleotides used in recombineering and in vitro SSA tests are described in Table 2. The purified RecA and SSB proteins of E. coli were purchased from Tebu.

PSI-Blast search

HkaK protein sequence from HK620 (annotated later in the text by SSB HK620 ) and Orf31 protein sequence from PA73 (annotated later in the text by SSB PA73 ) were compared by PSI-BLAST against the NRDB (non redundant data base) with standard parameters. These comparisons permitted to link the orf31 and hkaK genes to atypical two OB-fold SSB proteins from some phages infecting

Staphylococcus aureus.

Recombineering in the cat gene

To select recombinants, an assay based on the acquisition of chloramphenicol resistance upon recombination was set up. For this, a cat SS gene with two stop codons separated by 6 nucleotides was constructed: insertion of T528G and C535T stop mutations into the cat gene of pACYC184 was done by site-directed mutagenesis. The E. coli thiF and thiE genes were replaced by this mutated gene in an AB1157 recA mutS background (see Suppl. Text for details for the recombineering selection choice, and for strain constructions). To test easily various phage recombinase genes, a plasmid expression system derived from the thermosensitive, low copy number plasmid pKD46 was used. The plasmids carrying recombinases under the control of an arabinose inducible promoter, with or without accessory factors, were transformed into this strain.

An over-night culture grown at 30°C was diluted at the hundredth in 50mL of SLB+ampicillin (35 g/L Bactotryptone, 20 g/L yeast extract, 80 mM NaCl and 5 mM NaOH, 100µg/mL ampicillin), supplemented with 0.1 M of MOPS to maintain the pH at 7 all along the cell growth, and grown at 30°C until an OD 600nm of 0.2. The recombinases and their partner were induced by addition of arabinose to a final concentration of 0.2% and the culture was shifted to 37°C. After 45 minutes, the growth was stopped by chilling in an ice-cold bath (OD 600nm between 0.6 and 1). The cells were centrifuged in a pre-cooled centrifuge for 7 minutes at 4°C. Cells were resuspended in 50 mL of cooled glycerol 10% and centrifuged again. They were washed in 1 mL of cold glycerol 10% in a precold 1.5 mL tube twice, and then resuspended in 300 µL of cold glycerol 10%.

Electroporation was performed with 1µg of 81 nt-long oligonucleotide gently mixed with 100 µL of prepared cells and electroporated at 2.5 V, with a resistance of 200 Ω and a capacitance of 25 µFd. 900 µL of SOC media was added and electroporated cells were grown for 1 h at 37°C. Dilutions (done in SOC media) were then mixed to 5 mL of heated Top Agar 7 (10 g/L Bactotryptone, 2.5 g/L NaCl and 7 g/L Agar) and plated on LB agar to count total viable cells (titers of ~10 9 cells/ml are obtained, with this "top layer" method, which allows the best recovery of electroporated bacteria). To count CmR recombinants, the same procedure was used, except that after 2 h of growth in the top-agar layer at 37°C, a second top-layer of a mix of 5 mL of heated Top Agar 7 supplemented with 60 µg/mL of chloramphenicol was added. Viable cells were numerated after 24h of growth at 37°C, and recombinant cells were numerated after 48 h. The frequency of recombination was established by the ratio recombinant cells divided by viable cells.

Protein purification

UvsX T4 , Redβ λ , Sak4 PA73 , SSB PA73 and SSB HK620 were purified from Lemo21-DE3 Escherichia coli transformed by pETM30-derived plasmids (see supplementary methods for plasmid construction details). Cells were cultured into 800mL of LB+kanamycin (30µg/mL) at 37°C until an OD 600nm of 1. The culture was then transferred at 20°C and the recombinases tagged by GST were induced by IPTG at a final concentration of 100µM. Cells were then cultured overnight at 20°C and harvested by centrifugation at 3500 rpm for 30 min at 4°C. They were resuspended at 4°C in solution A (100 mM Tris HCl pH7.5, 100mM NaCl, 5 mM MgCl 2 , 1 mM DTT, 10% Glycerol, triton 1X, 1 mM PMSF, 8 µg/mL aprotinine) for UvsX T4 , RedB λ , SSB PA73 and SSB HK620 constructions or solution B (100 mM Tris HCl pH8.5, 500 mM NaCl, 5 mM MgCl 2 , 1 mM DTT, 10% Glycerol, triton 1X, 1 mM PMSF, 8µg/mL aprotinine) for Sak4 PA73 construction. These solutions were supplemented by 2.5 mg/mL of lysosyme and stirred on ice for 15 min. Cells were then lysed by sonication using a vibracell 75042 sonicator at 25, 35, 45, and 55 % intensity (1 min at each intensity). The lysate was then clarified by centrifugation (30 min at 20000 rpm at 4°C) and 5mL of GSH-agarose beads resuspended in buffer C (10 mM tris-HCl pH7.5, 5 mM MgCl 2 , 100mM NaCl, 1 mM DTT) was added. The mixture was incubated at 4°C for 1 h under agitation. The beads were then transferred on a gravity flow column (Biorad) and washed with 25 mL buffer D (10 mM tris-HCl pH7.5, 5 mM MgCl 2 , 1 M NaCl, 1 mM DTT). A second wash was done with 25 mL of buffer C. The fusion proteins were eluted with 15mL of a buffer E (10 mM tris-HCl pH7.5, 5 mM MgCl 2 , 150 mM NaCl, 1 mM DTT, 10 mM GSH) for UvsX T4 and RedB λ constructions, or buffer F (10 mM tris-HCl pH8.5, 5 mM MgCl 2 , 150 mM NaCl, 1 mM DTT, 10 mM GSH) for Sak4 PA73 , SSB PA73 and SSB HK620 constructions. Except for Sak4 PA73 , the his-GST-tag was removed by the addition of TEV protease (his-tagged) at a concentration of 1/100 (w/w) overnight at 4°C. The his-GST-tag and his-TEV protease where then removed using a His-trap fast flow 5mL column. The flow through containing the proteins was dialysed overnight at 4°C against buffer C. The elution containing the GST-Sak4 PA73 construction was dialysed overnight at 4°C against buffer D. SDS-PAGE was used to control each step of the purification process.

The protein concentrations were determined by the A 380nm , using the ϵ of each protein: 1.13 cm -1 M -1 for UvsX T4 , 1.45 cm -1 M -1 for RedB λ , 1.25 cm -1 M -1 for GST-Sak4 PA73 , 1.51 cm -1 M -1 for SSB PA73 and 1.17 cm -1 M -1 for SSB HK620 .

SSB gel shift assays

SSB proteins were incubated with the Cy5-labelled oligonucleotide GO47 (10 nM) for 15 minutes at 37°C in 10 µL of buffer G (10 mM tris-HCl pH7.5, 5 mM MgCl 2 , 1 mM DTT, 0.1 mg/mL BSA).

Saccharose to 8% final concentration was added prior to gel loading. Electrophoresis was performed with 10% acrylamide/bis-acrylamide (19:1) gels and 1X TBE migration buffer, at 120V for 2 hours, and the gel was revealed by the BioRad ChemiDoc TM MP imaging system.

ATPase assay

ATPase activity was assayed by linking ATP hydrolysis to NADH oxidation: in the presence of lactate deshydrogenase (LDH) and pyruvate kinase (PK), for each ATP molecule hydrolyzed, a NADH molecule is oxidized and the solution absorbance at 340nm decreases [START_REF] Pullman | Partial resolution of the enzymes catalyzing oxidative phosphorylation. I. Purification and properties of soluble dinitrophenolstimulated adenosine triphosphatase[END_REF]. The reactions were carried out, with or without 10 nM of GO34, at 37°C in a 70 µL final volume of Buffer H (10 mM Tris-HCl pH7.5, 5 mM MgCl 2 , 1 mM DTT, 0.1 mg/mL BSA, 7.5 mM phosphoenol pyruvate, 0.8mM NADH, 1mM ATP) with 1 µL PK/LDH mix from Sigma-Aldrich ® . The recombinases were used at 0.5 µM for RecA E.coli , 2 µM for Redβ λ , 0.5 µM for UvsX T4 , 0.5 µM for GST-Sak4 PA73 . Aliquots of 10µL were taken at different times and mixed in 40µL of 25mM EDTA to stop the reaction. The A 340nm was measured in 96 wells plates by Tecan Infinite ® M200 reader. The concentration of NADH was determined as a function of absorbance, using a standard NADH curve.

Single strand annealing assay

The Cy5-labelled oligonucleotide GO47 (10 nM) was preincubated with one recombinase (0.5 µM for RecA E.coli , 2 µM for Redβ λ , 0.5 µM for UvsX T4 , 0.5 µM for GST-Sak4 PA73 ) for 2 minutes at 30°C in 20µL of final volume of the buffer G (10 mM tris-HCl pH7.5, 5 mM MgCl 2 , 1 mM DTT, 0.1 mg/mL BSA). The reaction was started by the addition of one more or less diverged oligonucleotide (10 nM, see Table2). After 7 minutes, 4.5µL of the reaction was mixed with 3µL of stop buffer (by 10 mM of tris-HCl pH7.5, 3 µM of GO35 (untagged GO47), 0.5% of SDS and 0.2 mg/ml of proteinase K), and incubated 5 minutes at 30°C. The sample was loaded on a 10% acrylamide/bis-acrylamide (19:1) gel with a 1X TBE migration buffer. Electropheresis was performed at 120V for 2 hours, and the gel was revealed by the BioRad ChemiDoc TM MP imaging system. For kinetics, the same protocol is repeated in 50µL of reaction volume.

Strand exchange assay

Circular ssDNA from ΦX174 (10 µM in nucleotide) was incubated with 5 µM for RecA E.coli , or 1.16 µM for GST-Sak4 PA73 , or both, for 10 min at 37°C in 8 µL of final volume of the buffer H (10 mM Tris-HCl pH7.5, 5 mM MgCl 2 , 1 mM DTT, 0.1 mg/mL BSA, 1 mM ATP, 20 mM phosphocreatin and 10 units of creatin kinase). A second step of incubation with RecA E.coli or GST-Sak4 PA73 was added for 1 minute at 37°C in certain reactions, indicated in the figure legend. SSB was added (1 µL for a final concentration of 0.5 µM for SSB E.coli or 1 µM for SSB PA73 ; milliQ water was added when there was no SSB) to the solution. The reaction was kept at 37°C for 10 min. Strand exchange reaction was started by addition of 1µL containing PstI-linearized double strand ΦX174 was added for a final concentration of 10 µM (in nucleotides). After 30 minutes incubation at 37°C, the reaction was stopped by adding 2.5 µL of SI stop buffer (150 mM EDTA, 6% of SDS and 0.8 mg/ml of proteinase K). After at least 15 minutes at 37°C, the sample was loaded on a 0.8% GTG-agarose gel with a 1X TAE migration buffer.

Electrophoresis was run at 100 V for 5 hours at 4°C. The gel was colored by Ethidium Bromide and revealed by the BioRad ChemiDoc TM MP imaging system.

RESULTS

Temperate phages encoding Sak4 often encode an atypical SSB nearby

HK620 is the only sequenced phage infecting E. coli which encodes a Sak4. In the original annotation of its genome (Clark et al., 2001), four predicted genes were suspected to participate to homologous recombination by comparison with the Salmonella typhimurium phage P22 genome organization, as phage genomes are often organized into functional modules. These genes were therefore initially annotated as arf, erf, abc1 and abc2 respectively, similarly to P22. A map of this region in the two phages is shown Fig. 1. Upon closer inspection, only one of these four genes shares homology with a P22 gene: abc2. The arf HK620 gene is orfan, and has no homology to arf of P22, we therefore name it hkaM HK620 . The erf HK620 gene is not homologous erf P22 , but similar to the new family of recombinases exemplified by sak4 of phage Φ31, a core-only RecA-like recombinase (Lopes et al., 2010). Below, it will therefore be designated as sak4 HK620 . Finally, the abc1 HK620 gene has no similarity to abc1 P22 , despite occupying a similar genetic position in the recombination module. A PSI-Blast analysis indicated rather homology with a family of SSB-like proteins, which have the particularity to contain two OB-folds. This family encompasses members from various origins (phages, Deinococcus radiodurans, Archaea) (see methods). Some of the SSB detected by PSI-Blast were nevertheless shorter in length, and contained only one OB-fold. Interestingly, all these SSB orthologs of phage origin happen to be placed next to a sak4 gene (Suppl. Fig. 1). However, the reverse is not true, only 80% of the sak4 genes were found next to a gene encoding an SSB (suppl. Table 3). We renamed therefore abc1 HK620 as ssb HK620 . Finally, the abc2 HK620 gene shares 94% identical amino-acids with abc2 P22 (Clark et al., 2001), which functions as a hijacker of RecBCD E.coli , converting the potent ds exonuclease into a 5'-3' exonuclease which prepares DNA extremities for recombination (Murphy, 2000).

We searched for other 'local co-occurrences' among the 44 predicted sak4 genes: phage HK620 was the only one to possess an abc2 gene (2%), but 25% had a recE-like exonuclease (an accessory factor), which prepares singlestranded substrates for the RecT recombinase ( (Kuzminov, 1999) for review), and the remaining 73% had no detectable equivalent function.

We therefore undertook the study of two Sak4

in parallel: the one encoded by HK620, and one present on PA73, a phage infecting Pseudomonas aeruginosa (recE accessory factor, see Fig. 1). In both phages, a gene encoding a putative single strand binding protein was present next to the sak4 gene (fig. 1), and given the preferred association of these two genes detected above, the study involved these SSB as well. In bacteria, the SSB protein is ubiquitous and essential: the gene cannot be deleted. Its main function is to facilitate the replication process, but it also has a role in homologous recombination: SSB E.coli protects the ssDNA from RecA E.coli nucleation, but once RecA E.coli is nucleated on DNA, SSB E.coli facilitates its polymerization by suppressing DNA secondary structures. In addition, it is supposed that SSB E.coli catches the displaced strand during the D-loop formation, to stabilize the recombination intermediate [START_REF] Cox | Motoring along with the bacterial RecA protein[END_REF][START_REF] Shereda | SSB as an organizer/mobilizer of genome maintenance complexes[END_REF]. The reason why phages encode their own SSB proteins is not clear at present. Recently, it was shown for phage SPP1 that its SSB intoxicates the bacterial replication fork [START_REF] Lo Piano | Recombination-dependent concatemeric viral DNA replication[END_REF]. Here, we investigated whether phage SSB also plays a role in phage recombination. infecting Salmonella typhimurium, has four genes in operon involved in homologous recombination: arf P22 of unknown function, erf P22 encoding a Rad52-like recombinase (Lopes et al., 2010), abc1 P22 and abc2 P22 , producing RecBCD E.coli hijackers. HK620, infecting E. coli, also has four genes potentially involved in homologous recombination (Clark et al., 2001): hkaM HK620 of unknown function, sak4 HK620 , encoding a core-only Rad51-like recombinase (Lopes et al., 2010), ssb HK620 , encoding a single strand binding protein, and abc2 HK620 , homologous to abc2 P22 . In the Pseudomonas aeruginosa phage PA73, the two genes flanking sak4 PA73 are ssb PA73 , homologous to ssb HK620 , and recE PA73 , encoding an exonuclease. In the E. coli phage λ the recombination module contains four genes: gam λ , encoding an inhibitor of RecBCD E.coli , redβ λ , homologous to erf P22 (Lopes et al., 2010), redα λ , encoding an exonuclease, and orf60a λ of unknown function.

The SSB HK620 is essential for Sak4 HK620 SSA activity in vivo

To test the capacity of Sak4 recombinases to anneal ssDNA in vivo, alone or in combination with potential cofactors, a recombineering assay was performed: the Maj98 oligonucleotide, an 81-mer complementary to the mutated cat SS gene placed into the E. coli bacterial chromosome, was designed such that it could pair with the lagging strand, relative to the replication fork. Upon successful annealing, the chloramphenicol resistance is reestablished (Fig. 2A, (Li et al., 2013)).

Because we also planned to test the tolerance of the recombineering reaction to mismatches, the mismatch repair gene mutS of the strain to be tested was invalidated. To decrease the background level of spontaneous mutants, that is high in such mutS strains, the cat SS gene had two mutations placed next to each other to be restored for chloramphenicol resistance (CmR) reestablishment. The resulting strain was transformed with different plasmids coding for recombinases with or without their accessory factors (see methods). In this strain the spontaneous occurrence of CmR revertants was below the detection level (10 -9 ). However, a mock experiment of transformation with the Maj98 oligonucleotide in the strain carrying the vector plasmid devoid of recombinase, pJA4, indicated that spontaneous SSA occurs in vivo, at a frequency of 1.2x10 -8 .

In a RecA + E. coli strain, the SSA remains at the same level as the spontaneous annealing. Indeed, it is known that RecA is not able to promote such annealing in vivo (Lopes et al., 2010). Among the many mutants that have been tested in an attempt to overcome the barrier to recombineering in E. coli, uvrD has not been tested, to our knowledge. As the deletion of this helicase is known to increase the recombination rate of RecA in classical conjugation assay (Veaute et al., 2005), we performed the recombineering experiment in an uvrD mutant, and observed no increase in recombination efficiency (Fig. 2B, 1 st panel).

We performed all following experiments in a recA background, to exclude any potential interference of RecA in the assay. In the presence of a pJA4 derivative expressing Redβ λ , SSA was increased by a factor of 80, relative to the spontaneous annealing. next, the pKD46 plasmid encoding the redβ λ module was tested (Datsenko and Wanner, 2000), Fig. 1, last map): in addition to the gene encoding Redβ λ , it contains the genes for Redα λ , a dsDNA-dependent 3'-5' exonuclease [START_REF] Subramanian | The enzymatic basis of processivity in lambda exonuclease[END_REF], Gam λ , an inhibitor of the exonuclease RecBCD E.coli (Court et al., 2007;[START_REF] Murphy | The lambda Gam protein inhibits RecBCD binding to dsDNA ends[END_REF] and Orf60a λ , of unknown function, but reported to enhance dsDNA-recombeening (Datsenko and Wanner, 2000). SSA was increased by a factor of 5 over the Redβ λ annealing (Fig. 2B, last panel).

In contrast to Redβ λ , Sak4 HK620 did not promote SSA by itself (Fig. 2B, 2 nd panel). To test whether Sak4 required an accessory protein, a plasmid encoding all three surrounding genes (ssb HK620 , abc2 HK620 and hkaM HK620 , see Fig. 1) was tested. This time, a frequency of recombinants 240-fold over the spontaneous annealing was obtained. We next investigated which gene was implied in this effect. The abc2 HK620 and ssbHK620 genes were tested separately. Abc2 HK620 had a very small effect on Sak4 HK620 -dependent SSA, but SSB HK620 restored almost completely the phenotype observed with the all three surrounding genes, whereas this protein alone did not increase the spontaneous SSA level.

We conclude therefore that in vivo, Sak4-promoted SSA annealing depends completely on the presence of its companion SSB.

Whereas Sak4 PA73 was found to promote SSA three fold above spontaneous annealing with a different recombineering assay (Lopes et al., 2010), it did not promote SSA in the present assay (Fig. 2B, 3 rd panel). A construction with two sak4 PA73 neighboring genes, ssb PA73 and recE PA73 (Fig 1 ) could not be performed, as it was toxic to E. coli, but a plasmid containing sak4 PA73 and ssb PA73 was obtained. No SSA was observed with this construction. We cannot exclude that these proteins of a Pseudomonas phage are inactive in the heterologous host E. coli.

Both Sak4 HK620 and Redβ λ anneal up to 12% diverged sequences in vivo

The capacity of the Sak4 HK620 recombinase to anneal diverged sequences was tested in vivo, using several 81 nt-long oligonucleotides with equally spaced mismatches (divergence from 2% to 28%), as presented in Table 2. Redβ λ was tested in parallel, as this protein is already known to tolerate up to 12-16% of divergence during recombination in vivo (Martinsohn et al., 2008;Li et al., 2013;[START_REF] De Paepe | Temperate phages acquire DNA from defective prophages by relaxed homologous recombination: the role of Rad52-like recombinases[END_REF]. Both Redβ λ and Sak4 HK620 were tested together with all their accessory factors, given their high frequency of SSA (Fig. 2B). Annealing efficiency was high for 2% and 6% diverged pairs, and decreased sharply for 12% of divergence (Fig. 2C). No recombinants were detected for 16% diverged sequences. Interestingly, the two recombinases were similarly sensitive to divergence in vivo, suggesting that some common factor limits the integration of highly diverged pairs, such as those formed in vitro between 20% diverged oligonucleotides. This limiting factor may be exonuclease degradation of the oligonucleotides prior to their annealing, so that final duplexes are shorter and therefore less stable. Indeed, sequencing of 8 and 9 recombinants for the Redβ λ and Sak4 HK620 assays respectively, among those obtained with 12% diverged sequences, showed that all but one had integrated less than the total length of the input oligonucleotide (Suppl. Fig. 2). A slight tendency of a preferred degradation of the 3' end of the oligonucleotide was observed: 80% of integrated oligonucleotides were degraded in 3' relative to the incoming oligonucleotide, and 65% in 5' (Suppl. Fig. 2). A previous study from the Court laboratory has shown that the degradation in 5' is due to the exonuclease activity of Pol I and degradation in 3' to ssDNA exonucleases (Li et al., 2013).

Sak4 proteins are hardly soluble

To further study the Sak4 activity in vitro, Sak4 HK620 and Sak4 PA73 were expressed in E. coli with a GST tag (see Methods). The GST-Sak4 HK620 fusion formed inclusion bodies and was not studied further in vitro. The GST-Sak4 PA73 fusion was more soluble, but precipitated upon GST tag removal. It was therefore characterized as a fusion protein below (Fig. 3, lane GST-Sak4 PA73 , the two lower migrating bands are heat shock proteins, and the small MW band is GST). We investigated the biochemical properties of the purified fusion, hereafter designated as GST-Sak4 PA73 . In addition to Sak4, two well characterized phage recombinases were included in this study, as a comparison: UvsX T4 and Redβ λ .

They were also expressed as GST tagged fusion, remained soluble upon tag removal, and were therefore studied in their untagged form (Fig. 3). Finally, a commercial preparation of RecA E.coli was also included in the assays (Fig. 3).

The phage-encoded SSB-like proteins have affinity for ssDNA

As the activity of Sak4 is dependent on its cognate SSB in vivo, SSB HK620 and SSB PA73 were purified as well, and compared to SSB E.coli

. The two phage SSB proteins were produced as GST-tagged fusion, and remained soluble upon GST removal (Fig. 3, two last lanes, SSB HK620 is shorter in size and corresponds to a single-OB fold version of phage SSB). In order to validate the predicted single strand binding function, increasing concentrations of each SSB were incubated in the presence of Cy5 labelled, 81-nt long oligonucleotide (GO47), and the products were separated by electrophoresis. Each phage SSB shifted the ssDNA on gel. Based on the intensity of the non-displaced band, corresponding to the free oligonucleotide, an 8-fold reduced affinity was observed, compared to SSB E.coli (Fig. 4). The shifted bands were of a much lower mobility in the case of SSB PA73 , probably proportional to the protein size.

We next tested whether GST-Sak4 PA73 and its cognate SSB interacted, by GST pull-down, and did not find any evidence of a direct interaction. No indication of any interaction between GST-Sak4 PA73 and SSB E.coli was found either. Redβ λ , UvsX T4 , Sak4 PA73 , SSB PA73 and SSB HK620 were purified as described in the methods section, RecA E.coli was from Tebu. The molecular weights (MW) of the ladder are indicated.

GST-Sak4 PA73 does not degrade ATP

As Sak4 PA73 has Walker A and B ATP-binding sites, we investigated its capacity to hydrolyze ATP. ATP hydrolysis was monitored using a coupled spectrophotometric assay (see Methods). RecA E.coli and UvsX T4 hydrolyzed ATP in the presence of the 81nt long GO34 ssDNA, whereas Redβ λ did not, as expected.

Interestingly, GST-Sak4 PA73 did not hydrolyze ATP either (Fig. 5).

GST-Sak4

GST-Sak4

GST-Sak4

GST-Sak4 (ssc, Fig. 6A). For RecA E.coli , the reaction is ATP-dependent [START_REF] Rehrauer | Alteration of the nucleoside triphosphate (NTP) catalytic domain within Escherichia coli recA protein attenuates NTP hydrolysis but not joint molecule formation[END_REF]Rice et al., 2001), and stimulated by SSB (Kowalczykowski and Krupp, 1987). We first tested RecA E.coli and GST-Sak4 PA73 in the presence and absence of SSB E.coli (Fig. 6B and6C). RecA E.coli at low concentration (1µM, Fig. 6B) did not promote SE unless SSB E.coli , was added, in which case joint molecules (JM) were formed. At a higher concentration (2µM, Fig. 6C), RecA 2012). So, we tested whether GST-Sak4 PA73 could stimulate RecA E.coli , in the 1-2µm range, where RecA E.coli is stimulated by SSB E.coli . No Sak4-dependent stimulation of the SE was observed, regardless of the order of addition of the two proteins (Fig. 6B and 6C, lanes 7-9). In fact, the presence of GST-Sak4 PA73 inhibited SE, at 2µM of RecA E.coli (Fig. 6C, compare lane 2 and 7-8). The experiment was also performed in the presence of SSB E.coli , and again no stimulatory effect of GST-Sak4 PA73 was found. Its inhibitory effect was lessened by the presence of SSB E.coli , and observed only when GST-Sak4 PA73 was added prior to RecA E.coli (Fig. 6C, compare lanes 10 and 11, a simultaneous addition, lane12, has intermediate effect). Finally, the whole experiment was repeated with SSB PA73 instead of SSB E.coli , for the 2µM RecA reactions. SSB PA73 did not stimulate RecA E.coli , nor did it permit to unveil any SE activity for GST-Sak4 PA73 (Fig. 6D).

RecA E.coli ssc dsl jm nc ssl A B SSB E.coli - - -+ + + - - -+ + + -+ - -+ -1 2 + 1 2 + - - + - -+ 2 1 + 2 1 + C - - -+ + + - - -+ + + -+ - -+ -1 2 + 1 2 + - - + - -+ 2 1 + 2 1 + - - -+ + + - - -+ + + -+ - -+ -1 2 + 1 2 + - - + - -+ 2

GST-Sak4 PA73 performs SSA in vitro, and anneals up to 20% diverged single stranded DNAs

Finally, the SSA activity of GST-Sak4 was monitored, by incubating it in the presence of two complementary 81 nt long oligonucleotides, followed by protein removal and analysis of the DNA products on acrylamide gels. Kinetics of recombinasepromoted SSA is shown Fig. 7, compared to spontaneous annealing. GST-Sak4 performed efficient and rapid annealing in vitro, as ~100% of the dsDNA product was formed within the first minute of incubation, whereas spontaneous annealing had not occurred yet at this time point, and appeared slowly over time, with 20% ds products being made at the 30 minutes point.

To test the effect of divergence on the efficiency of annealing, increasingly diverged oligonucleotides were next used (0 to 28% divergence, the sequence of the various 81 nt-long nucleotides are shown in Table 2). GST-Sak4 was compared to RecA and, as a negative control, the GST protein was tested. The results of Fig.

8A and 8B are shown at a single time point of 7 min. No activity above the level of spontaneous annealing (10%) was observed with GST. Both GST-Sak4 PA73 and RecA E.coli promoted SSA, up to 20% diverged sequences. These two recombinases have therefore an efficiency of annealing comparable to that observed for Redβ λ up to 20% diverged sequences (see Suppl. Fig. 3). Interestingly, Redβ λ also annealed 28% diverged sequences, but the dsDNA product had a mobility in gel lower than expected (Suppl. Fig. 3). We have no hint at the nature of this shifted band at present, it may be due to partial melting of this highly diverged duplex DNA. We noted a slight, but reproducible increase of the Redβ λ SSA efficiency when divergence was increased, up to 20% (Suppl. Fig. 3). We have no explanation for this tendency. Contrary to GST-Sak4 PA73 and RecA E.coli , UvsX T4 did not have any SSA activity in the absence of ATP (Suppl. Fig. 3). This fits with its dependence on ATP to bind ssDNA [START_REF] Maher | Coordinated Binding of Single-Stranded and Double-Stranded DNA by UvsX Recombinase[END_REF].

The same SSA assay was repeated in the presence of 1mM ATP (Fig. 8C and 8D, Suppl Fig. 4). UvsX T4 performed SSA with oligonucleotides up to 28% divergence this time (Suppl. Fig. 4). In contrast, the efficiency of SSA was unchanged by the presence of ATP for GST-Sak4 PA73 , RecA E.coli and Redβ λ, except that RecA E.coli was active also on 28% diverged sequences.

We next asked whether the host, and/or the phage encoded SSB proteins would inhibit the various recombinase's SSA activity. We first verified that none of the three SSB performed SSA in vitro (Suppl. Fig. 5). Increased concentrations of SSB E.coli or SSB PA73 were incubated with recombinases and oligonucleotide GO47 for 2 minutes before the addition of the complementary oligonucleotide GO34 (Suppl. Fig. 6). Remarkably, the two SSB had completely different effects on the SSA reactions: SSB PA73 did not inhibit the SSA of any recombinase, whereas SSB E.coli inhibited the SSA activity of RecA E.coli , GST-Sak4 PA73 and UvsX T4 in a dose dependent manner, with complete inhibition achieved at 0.5µM of SSB E.coli , corresponding to SSB:recombinase molar ratios of 1:1 (as monomers). The Redβ λ activity was only partially inhibited at the highest SSB concentrations tested, which corresponded to SSB E.coli /Redβ λ ratio of 1:4 (as monomers). In vivo, both SSB should co-occur in the host during a phage cycle, we therefore tested the effect of a mix of both SSB proteins on the SSA activity: using a 250µM concentration of SSB E.coli and 8-fold more of SSB PA73 , the SSA activity of both RecA E.coli and GST-Sak4 PA73 , were inhibited. This suggests a dominance of SSB E.coli over SSB PA73 , which may be related to its higher affinity for ssDNA (Fig. 4).

GST RecA E.coli GST-Sak4 PA73 RecA E.coli GST-Sak4 PA73 GST RecA E.coli GST-Sak4 PA73

DISCUSSION

Sak4 has SSA activity

The Sak4 protein, despite sharing homology with RecA, behaves more like a phage recombinase of the Rad52 superfamily, both in vitro and in vivo. Technical reasons prevented the parallel in vivo and in vitro study of a single Sak4, but the results reported here for Sak4 HK620 in vivo and Sak4 PA73 in vitro are consistent. Sak4 HK620 promoted SSA in vivo, in a recombineering assay, to levels comparable to Redβ λ , provided its companion SSB was co-expressed. The only form of Sak4 that was soluble in vitro was the GST-Sak4 PA73 fusion, so it was investigated further. It exhibited an SSA activity, but no strand exchange activity, like Redβ λ . We cannot exclude that the GST fusion prevented the observation of the strand exchange activity. Furthermore, despite the clear presence of Walker A and B motifs in both Sak4, no ATPase activity was detected for GST-Sak4 PA73 . This may suggest that the main Sak4 activity is not strand-invasion mediated recombination, like RecA, but SSA. This SSA would occur without degrading ATP, as is also observed for Rad52-like proteins.

Interestingly Sak4 HK620 needs the presence of its cognate SSB protein in vivo to carry out recombineering. This SSB dependence is not observed in vitro in SSA assays with GST-Sak4 PA73 . But no inhibition is observed either, contrary to what is seen when the host SSB protein is added into SSA reactions. One may imagine a scenario where phage-encoded SSB are present at the replication fork, and do not shield ssDNA from the phage recombinase. Otherwise, it is possible that SSB HK620 prevents the inhibition by an unknown factor that would inhibit Sak4 HK620 -mediated SSA (see below). Contrary to GST-Sak4 PA73 , we noticed that the SSA mediated by Redβ λ was not completely inhibited by SSB E.coli on linear substrates (Suppl. Fig. 6). It was previously shown that Redβ λ anneals ssDNA in the presence of SSB E.coli at low concentration of DNA [START_REF] Muniyappa | The homologous recombination system of phage lambda. Pairing activities of beta protein[END_REF]. It may be that Redβ λ is loaded first, before SSB E.coli , as is supposedly the case if it interacts with its cognate exonuclease Redα λ .

In humans, the core-only RecA XRCC2 protein is thought to act essentially as a recombination accessory factor. Indeed, when present in a complex together with RAD51BCD, it promotes SSA in vitro (Yokoyama et al., 2004). We found no evidence of any interaction between Sak4 and RecA in vivo, as SSA occurred at similar levels in RecA + or recA-deleted strains (not shown). The function of the archaeal RadB protein is not well understood at present, but it presents some activities similar to Sak4. It inhibits the SE activity of RadA, as we found for GST-Sak4 PA73 , when added onto ssDNA prior to RecA E.coli in the reaction. Furthermore, RadB does not degrade ATP, but it binds to it, and hydrolyses without releasing it (Komori et al., 2000). It is possible that Sak4, in the same manner, binds to ATP but does not release it.

Sak4 being essentially truncated for its C-terminal domain, relative to RecA, it may behave as partially deleted RecA mutants. The removal of amino acids from the C-terminal end of RecA E.coli leads to improved SE reactions at alkaline pH (Lusetti et al., 2003). But at pH7, the most C-terminus deleted RecA E.coli no longer promotes SE (Lusetti et al., 2003). This may explain the SE incapacity of Sak4.

Indeed, this domain of RecA is thought to be important for stabilizing the three-strands intermediate (De Vlaminck et al., 2012).

The in vitro SSA activity of RecA E.coli is inhibited in vivo

We report here that RecA E.coli promotes SSA in vitro. This was the first demonstrated recombination activity of this recombinase, deduced from nitrocellulose binding assays (Bryant and Lehman, 1985).

No trace of SSA activity for RecA E.coli is nevertheless found in vivo, in a recombineering assay ((Lopes et al., 2010) and this work). It is usually considered that the high affinity of SSB E.coli for ssDNA shields the substrate for RecA E.coli , as we saw effectively in vitro. Still, one may have expected that RecO E.coli , through its mediator role, would remove SSB E.coli to insert RecA E.coli (Morimatsu and Kowalczykowski, 2003;Handa et al., 2009). Moreover, a recent work in single molecules (Bell et al., 2012) filament (Veaute et al., 2005). But even in an uvrD mutant, RecA E.coli is prevented from annealing oligonucleotides at the replication fork (Fig. 2B). At present, we have no clue therefore on the reason why RecA E.coli does not perform recombineering in vivo.

We found in addition a remarkable tolerance of RecA E.coli to mismatches in vitro, as it annealed oligonucleotides differing by up to 28% of divergence. The tolerance of RecA E.coli to mismatches has already been observed both in strand invasion assays on 13% diverged substrates, i.e. 2 mismatches over a 15 bp heteroduplex (Malkov et al., 1997), and a strand exchange assay between M13 and fd molecules [START_REF] Worth | Mismatch repair proteins MutS and MutL inhibit RecA-catalyzed strand transfer between diverged DNAs[END_REF]Tham et al., 2013), but strand invasion and strand exchanges reactions are different, and probably more complex than SSA.

Redβ λ and GST-Sak4 PA73 tolerate the presence of mismatches during SSA, both in vitro and in vivo

We reported earlier that several Rad52-like phage recombinases recombine diverged sequences in vivo, both during DNA inversion on a phage substrate (Martinsohn et al., 2008), and during exchanges between chromosomal and phage sequences [START_REF] De Paepe | Temperate phages acquire DNA from defective prophages by relaxed homologous recombination: the role of Rad52-like recombinases[END_REF]. In addition, Redβ λdependent recombineering was reported to proceed with diverged oligonucleotides up to 17% (Li et al., 2013). Here, we found in vivo annealing for Redβ λ up to 12% diverged sequences, but no longer at 16% (the difference may lie in the different locus used for recombineering, compared to the Li et al.

study (Li et al., 2013)). Interestingly, Sak4 HK620 behaved in a way similar to Redβ λ , with respect to divergence in vivo. In vitro, however, it could not pair 28% diverged oligonucleotides, contrary to Redβ λ . Overall, the tolerance to mismatches in vivo seems therefore a property shared by two distinct families of phage-encoded recombinases.

As mentioned in the introduction, phages encode three recombinase superfamilies (Lopes et al., 2010). The first and most populated one is the Rad52-like, comprising the Redβ/RecT, Erf and Sak families, in which some members have recognized SSA activity (Kmiec and Holloman, 1981;Datta et al., 2008;Ploquin et al., 2008). The same holds true for the second, Gp2.5 family (Lopes et al., 2010).

Here we report that members of the third family of Rad51-like recombinases, UvsX T4 , Sak4 PA73 , but also RecA E.coli itself, are capable of SSA. Furthermore, it is known that some Rad51 orthologues in Eucaryota and Archaea promote SSA and not SI: this is the case of the BCDX2 complex (Yokoyama et al., 2004), composed by Rad51BCD and the core-only XRCC2, and for RadB of P. furiousous (Komori et al., 2000). It may be that the very first activity of recombination of the ancestral RecA was to simply anneal ssDNA, and this activity evolved to a strand invasion activity leading to the homologous recombination as we know it now.

Construction of MAC1802 strain

MAC1802 is a strain containing in its chromosome a mutated cat gene. Site directed mutagenesis, to introduce two stop codons separated by 6nt into the cat gene of pACYC184 (cat SS allele), was performed with the help of oligonucleotides J85 and J86 (supplementary table 1), that are complementary. The two stop codons generate G:A and T:G mispairs during the recombineering experiment using oligonucleotide Maj115, which has the polarity of a lagging strand. Oligonucleotides J84 and J87 flank the cat gene of pACYC184 ( 4) and overlap two BmgBI sites of this plasmid. The two halves of the mutated cat gene were amplified with the J84-85 and J86-87 pairs (652bp and 243bp fragments, respectively), and the full cat SS mutated gene was then produced using these two PCR fragments as initial primers, as well J84 and J87. The full gene fragment was then cloned into the BmgBI site of pKD4 (2). This cat SS mutated gene, together with the KanR marker of pKD4 were next introduced by recombineering into the thi operon (10kbp away from rpoB), in place of thiE and thiF, and in the same orientation, of strain JJC40 (AB1157 hsdR), using oligonucleotides Maj80 and Maj81. The resulting cassette was next transduced by P1 into AB1157 (strain MAC1716). The mutSΩ (specR) allele ( 5) was next introduced by P1 transduction in this strain (MAC1774), and finally the ΔrecA306 srl:Tn10 allele (from strain GY5902, a gift of Dr. S. Sommer), to give strain MAC1802.

Plasmid constructions

Constructions of all plasmid are described in supplementary Table 2. Unless otherwise stated, plasmids (column 1) were constructed by the cloning of a PCR product (columns 2 to 5) into a plasmid (column 6 and 7). The gene amplified is indicated in the second column. PCR were performed with the high fidelity polymerase Phusion, using the DNA matrix indicated in third column, and the oligonucleotides indicated in columns 4 and 5. The vector (column 6) and the PCR product are digested by the enzyme stipulated in column 7. Brief plasmid description in column 8.

Supplementary Table 2: principally encode Rad52-like recombinases (Martinsohn, Radman, Petit 2008). Interestingly, the prophages encoding a recombinase have more mosaic genomes than the others (Bobay, Rocha, Touchon 2013). These phage recombinases are grouped into three super-families: the Rad52-like, the Rad51-like and the Gp2.5-like (Lopes et al. 2010). Each of these families has its particularities and its restrictions, which are discussed below.

A) Rad52-like recombinases are permissive annealases.

The main activity of these recombinases is SSA, they cannot invade a dsDNA to search for homology, unless under particular conditions. The most studied recombinase of this super-family is Redβ, from the phage λ, which was used as a representative model during my thesis. We have shown that this recombinase, as well as other members of this family, recombines diverged sequences both in vivo and in vitro up to 20% of divergence ( (Martinsohn, Radman, Petit 2008;Li et al. 2013), Chapter II and III).

As its main activity is SSA, the generally accepted hypothesis is that its action resides at the replication fork. Indeed, the only ssDNA present in a bacterium and protected from degradation is the template of the lagging strand at the replication fork. In agreement with this hypothesis, when DnaG, the primase that polymerizes RNA primers for the Okazaki fragments, is depleted, the free ssDNA at the replication fork is longer and the efficiency of recombination by Redβ is higher [START_REF] Lajoie | Manipulating replisome dynamics to enhance lambda Red-mediated multiplex genome engineering[END_REF]. Furthermore, mutations into the gene encoding Pol I, the polymerase that degrades RNA primers to polymerize DNA, and Pol III, the polymerase that polymerizes DNA from primers, decrease the efficiency of Redβ mediated recombination (Li et al. 2013;[START_REF] Poteete | Involvement of DNA Replication Proteins in Phage Lambda Red-Mediated Homologous Recombination[END_REF] Another model is more attractive and fits with these results. First, the dsDNA has to be prepared for integration by being turned into a ssDNA complementary to the lagging strand template of DNA replication fork. Then, the ssDNA fragment can be integrated by annealing of its two extremities, regardless of the sequence in between (see Figure 15 in Chapter I, (Maresca et al. 2010)). To explain gene capture with this model, we need to suppose that the bacterial chromosome has been truncated into pieces of dsDNA, and then converted into ssDNA by exonuclease digestion. These pieces of ssDNA would then anneal at the replication fork by Redβ as in the Maresca et al. model.

How bacterial DNA is broken down to pieces during the phage replication is yet unknown.

However, this model is not sufficient to explain all our results. Indeed, the maximal length of the free ssDNA at the replication fork is equivalent to the length of Okazaki fragments, which measure between 1 and 2 kb, unless DnaG is mutated [START_REF] Tougu | The extreme C terminus of primase is required for interaction with DnaB at the replication fork[END_REF]. DNA fragments of the size of a gene, as antibiotic resistance genes in Chapter II, or smaller, as the predominant little mosaics observed by bioinformatics in Chapter II, are probably exchanged this way. However, in the promoter inversion model (Chapter II), the fragment is somewhat longer (1.5 kb to 2 kb) and may be difficult to recombine this way. Furthermore, an exonuclease has to digest 2 kb to obtain a ssDNA substrate sufficient to be annealed at the replication fork (as proposed in Maresca et al. model), and induce promoter inversion.

Redβ is also capable of promoting some level of SI. First, it promotes a certain type of strand exchange (Figure 19): if the oligonucleotide has paired partially to a single strand template, the Chapter IV: Discussion, Mosaic acquisition: causes and consequences, Phage recombinases, a major cause of mosaïcism 127 remaining duplex DNA is exchanged by the incoming oligonucleotide. This reaction has clear preference for a 5' first annealing [START_REF] Li | The beta protein of phage lambda promotes strand exchange[END_REF]. Second, Redβ permits strand invasion under certain circumstances: at low magnesium concentration, the DNA breathes by repulsion of negative charges of the two DNA strands and Redβ, as RecT, could find the complementary strand during the breathing (Noirot, Kolodner 1998;Rybalchenko et al. 2004). Such breathing exists when DNA is supercoiled, and Redβ probably finds homology this way when it is possible. Other enzymes facilitating dsDNA opening, such as helicases could also play a role. Maybe some accessory factors of Redβ remain to be found. In favour of a SI mechanism, some recombinants of the phage promotor inversion assay annalysed by Martinsohn et al. have symetrical end points in the inverted repeat (Martinsohn, Radman, Petit 2008). Indeed, the SI mechanism leads to a four branches Holliday junction that gives rise to symmetrical resolution products.

Another mechanism may be MHEJ. As discussed in the introduction (Chapter I), a phage Rad52 recombinase, RecT, promotes this type of events with a frequency of 2.10 -7 during phage induction [START_REF] Shiraishi | Roles of RecJ, RecO, and RecR in RecET-Mediated Illegitimate Recombination in Escherichia coli[END_REF][START_REF] Shiraishi | The role of UvrD in RecET-mediated illegitimate recombination in Escherichia coli[END_REF]. Furthermore, a group identified a major deletion (126 genes) in the bacterial chromosome of their E. coli strain during a recombineering experiment with

Redβ [START_REF] Vine | Detection by whole genome microarrays of a spontaneous 126-gene deletion during construction of a ytfE mutant: confirmation that a ytfE mutation results in loss of repair of iron-sulfur centres in proteins damaged by oxidative or nitrosative stress[END_REF]. The sequencing of the deletion point revealed that at the recombination junction, only 1 nt was identical. 53% identity on a 17 bp region surrounding the junction was present (unpublished results of the team, Figure 20). This observation lets us think that Redβ has the same ability as RecT, and probably more phage Rad52 recombinases. MHEJ may explain a part of the nonsymmetrical promoter inversion observed by Martinsohn et al. (Martinsohn, Radman, Petit 2008).

Redβ recombines diverged sequence, but to do so, it must (1) pair sequences with mismatches and

(2) resist to the MMR system. We saw in Chapter III that Redβ anneals DNA of sequences up to 20% of divergence as efficiently as perfectly identical DNA in vitro. The Court group analyzed deeper this ). The efficiency is retrieved when homologies are added at the oligonucleotide extremities.

However, the homologies seem to be more important in the 5' part than in the 3' part of the incoming oligonucleotide (Li et al. 2013). In the recombineering experiment, the Court group reported that MutS and MutL, of the MMR mechanism, inhibit Redβ mediated ssDNA recombineering (Costantino, Court 2003). Our group saw, in the phage promoter inversion assay, that MMR does not affect the Redβ mediated recombination (Martinsohn, Radman, Petit 2008). The difference between these two assays is the potential number of recombination events in bacteria at the same time. In recombineering, only one to four recombination templates (chromosomes) are available for recombination, so that at most 4 recombination events occur at the same moment. In Chapter IV: Discussion, Mosaic acquisition: causes and consequences, Phage recombinases, a major cause of mosaïcism 129 degrade ATP and to promote a complete strand exchange (Liu et al. 2011). Nevertheless, we saw in Chapter III that RecA and UvsX both anneal diverged olinucleotides in vitro. Tests cannot be performed with RecA for ssDNA recombineering on diverged oligonucleotides, because RecA does not anneal in vivo, but UvsX does, albeit with a low efficiency (Lopes et al. 2010). Some accessory factor may be needed to fulfill efficient SSA in vivo. Once such factors are identified, it will be interesting to test the fidelity of UvsX in this test.

Sak4 was never characterized until this study. We saw in Chapter III that it promotes SSA but not SI, under the conditions used. We cannot exclude that Sak4 devoid of its GST tag promotes SI.

Furthermore, Sak4 anneals diverged sequences both in vivo and in vitro. These capacities will be discussed deeper in paragraph II of this Chapter. Interestingly, Sak4 partially inhibits the activity of the host recombinase RecA in vitro. It could be interesting to test this inhibition in a classic UV assay with a RecA + E. coli strain carrying a plasmid expressing Sak4, with or without its SSB. If Sak4 inhibits RecA and does not promote SI, the UV induced DNA damages will not be repaired and the strain will have a UV sensitive phenotype.

SSB proteins are important for these recombinases. Indeed for the host recombinase RecA, SSB is necessary to promote efficient strand exchange ( (Kowalczykowski, Krupp 1987) and Chapter III), but it can be replaced by T4 SSB protein Gp32 in vitro [START_REF] Egner | Effects of various single-stranded-DNA-binding proteins on reactions promoted by RecA protein[END_REF]. Similarly, Gp32 is required for UvsX to promote efficient strand exchange. Furthermore, Gp32 presence enhances SSA and ATPase activities of UvsX [START_REF] Formosa | Purification and characterization of the T4 bacteriophage uvsX protein[END_REF][START_REF] Yonesaki | s'y répliquent et la lysent pour en sortir. Les phages dits tempérés font également des cycles lytiques, comme les virulents, mais peuvent aussi, après être entré dans la bactérie, s'intégrer dans son chromosome au lieu de se répliquer. On les appelle alors prophages. De temps en temps, les prophages sortent du cycle lysogène en s'excisant du chromosome suite à un stress et reprennent un cycle lytique. La structure des génomes des phages virulents s'approche de celle des génomes des bactéries : un squelette constant, émaillé de régions variables. Les génomes de phages tempérés par contre sont plus complexes. Lorsqu'on les compare[END_REF]. Finally, Sak4 does not anneal at the replication fork without its SSB protein (Chapter III). It is probable that these phage SSB proteins load onto ssDNA at the same time as E. coli SSB and permit Sak4 loading. To test this idea, UvsX may be expressed together with Gp32 to see if its recombineering activity is enhanced. Maybe in this precise case, the UvsY mediator will also be required.

C) Gp2.5-like recombinases are poorly explored

The Gp2.5 protein looks like an SSB because of its OB-fold, but has a supplementary α-helix (Hollis et al. 2001). Until recently (Lopes et al. 2010), Gp2.5 of T7 was the only one characterized protein of this family, it is therefore the most studied of this super-family. It promotes SSA both in vitro and in vivo [START_REF] Hyland | The DNA binding domain of the gene 2.5 singlestranded DNA-binding protein of bacteriophage T7[END_REF]Lopes et al. 2010). It also promotes SE in the presence of the phage helicase Gp4 in vitro [START_REF] Kong | Single-stranded DNA binding protein and DNA helicase of bacteriophage T7 mediate homologous DNA strand exchange[END_REF] As I presented in the introduction (Chapter I), the Herpes Simplex Virus (HSV) caries a gene encoding ICP8, which is distantly related to Gp2.5. It forms a 2-rings structure closing over one another as a box where two ssDNA are compared by sliding (Figure 16 in Chapter I, [START_REF] Tolun | Details of ssDNA annealing revealed by an HSV-1 ICP8-ssDNA binary complex[END_REF]). However, ICP8 shares with Gp2.5 only the middle segment of the protein, and it is not obvious that Gp2.5 has the same mode of action. The two quaternary structures of ICP8 and Gp2.5 should be compared to see if the ring structure could be found for Gp2.5.

2) Consequences on genomes

From a certain point a view, there is a tradeoff between fidelity and speed in the homologous recombination process. The relaxed fidelity of the phage recombinases permits to facilitate the exchanges between phages, but is damaging for the bacterial chromosome maintenance.

A) An advantage for phage genomes

As we saw above, all phage recombinases promote SSA in vivo, even UvsX, contrary to host recombinase RecA. Furthermore, most of them recombine diverged sequences (Gp2.5 unfaithful recombination activity is still to be demonstrated). These abilities change the way we imagine how phages protect their own genomes during replication compared to bacteria. Indeed homologous recombination permits bacteria to repair DNA damages with a high fidelity. In phages, recombinases probably repair faster their DNA at the expense of a lesser fidelity. Indeed, in vitro SSA between 81 nt long oligonucleotides is completed in less than a minute, even when sequences are diverged, whereas SI between 5 kb long DNA molecules is a slow process, needing some tens of minutes for completion and resolution. With SSA, phages can rapidly associate blocks of genomes on parts at least similar in term of homologies and functions to create as many viable phages as possible. This phenomenon separates clearly the way that phages stabilize their genomes, compared with bacteria.

It may be that fast and low fidelity repair is linked with phage mosaïcism, whereas slow and faithful repair is assiociated with backbone/variable segment (VS) structures found in Bacteria, because of the necessity for them to conserve their chromosome integrity.

To support this theory, Enterobacteria prophages encoding for a recombinase are more mosaic than others (Bobay, Rocha, Touchon 2013). The authors proposed that the phages that do not encode for a recombination system use that of the host. This point is discussed in our recently submitted review (Menoui, Hutinet et al., In Press, FEMS).

In competent bacteria, prophage recombinases promote recombineering [START_REF] Swingle | Oligonucleotide recombination in Gram-negative bacteria[END_REF]. They insert oligonucleotides at the replication fork that could bring punctual mutations into the bacterial chromosome. Larger fragments may also be incorporated and bring several mutations at a time corresponding to the microdiversity regions previously observed in the team [START_REF] Touzain | Small variable segments constitute a major type of diversity of bacterial genomes at the species level[END_REF].

More dangerous for the bacteria, major delections could occur, without apparent need for long homologous region as mentioned above for the MMET activity of Redβ [START_REF] Vine | Detection by whole genome microarrays of a spontaneous 126-gene deletion during construction of a ytfE mutant: confirmation that a ytfE mutation results in loss of repair of iron-sulfur centres in proteins damaged by oxidative or nitrosative stress[END_REF]).

Rad52-like recombinase genes are absent in bacterial genomes, except in prophages but then essentially repressed. The ability of these recombinases to recombine diverged sequences urges bacteria to counter-select their presence into their genomes, in order to maintain a faithful method for the repair of DNA breaks by the SI ability of RecA, and to protect from aberrant recombination mediated by Rad52-like recombinases. However, Eukaryote genomes succeeded to domesticate 3) A new definition of phage species?

Classification of phages into species as done for the three kingdoms of life is difficult because (1) there is no gene shared by all phages, as the 16S-rDNA (18S in Eukaryotes), and (2) phages are highly divergent. This is why it is necessary to consider another way to define species. Indeed, Animal species were first defined as capable of producing a viable offspring by sexual reproduction. The hidden process behind is homologous recombination. Indeed, in order to be able to reproduce, Eukaryotic cells need to align their homologous chromosome by a mechanism of homologous recombination. This means that the homologous recombination process determines the species boundaries. In the same manner, different bacterial species cannot exchange core genetic material because of the necessity of homologous recombination in the process. This process is highly regulated, by the ability of RecA in a first place, and then by the MMR system [START_REF] Stambuk | Mechanism and control of interspecies recombination in Escherichia coli. I. Mismatch repair, methylation, recombination and replication functions[END_REF]).

It may be convenient to consider a phage species definition based on homologous recombination.

The fact that their recombinases have a relaxed fidelity may change our vision of species for phages.

If the species membership can be circumscribed by the capacity to exchange DNA, two phages sharing a mosaic would belong to the same species. In the phage genomic tree, where phages are clusterised by their amount of shared genes [START_REF] Rohwer | The Phage Proteomic Tree: a Genome-Based Taxonomy for Phage[END_REF], the closer phages infect the same host, while most distant phages infect different hosts. It is thus possible that these close phages are in the same species. In the same way, in the reticulate classification proposed by the Toussaint group [START_REF] Lima-Mendez | Reticulate representation of evolutionary and functional relationships between phage genomes[END_REF], each cluster including phages with high levels of shared genes infects related bacterial species and a cluster could be considered as a phage species. A clear example of phage species is T4-like phages. Their genome are so close from each other that the species definition is clearly visible by full genome alignments (Ignacio-Espinoza, Sullivan 2012).

However, this way of thinking presents some difficulties. Indeed, in Chapter II we saw that active temperate E. coli phages are divided into two groups, probably two species, which do not exchange genome parts with each other: λ-likes and P2/Mu-likes. However when we look at prophages of these two "species", they exchanged genome parts with each other. This probably means that phages of these two "species" might cross with each other. The resulting offspring might be insufficiently viable, so this case is found only in defective prophages.

Chapter IV: Discussion, The phage core-only RecA Sak4…, … compared with RecA 133 II) The phage core-only RecA Sak4…

Sak4 has the particularity to share only one domain with RecA: the ATPase and DNA-binding domain.

Below, Sak4 will be first compared with RecA and then with other core-only RecA.

1) … compared with RecA

In vitro, both RecA and GST-Sak4 promote SSA and both do it between diverged oligonucleotides.

However, in vivo RecA cannot promote SSA while Sak4 does, but only in presence of its SSB. GST-Sak4 differs from RecA on other important aspects. Indeed, RecA hydrolyses ATP in a ssDNA-dependent way, while Sak4 does not. Whether it binds ATP without hydrolyzing it remains to be established.

Finally, GST-Sak4 cannot promote SI as RecA does (Chapter III). A trivial explanation could be that GST-Sak4 is not functioning like Sak4. Further efforts of purification are needed to obtain a soluble Sak4. The differences between these two proteins reside into the Ct ssDNA-binding supplementary domain of RecA. In a recent model, this domain is supposed to participate actively to the SE process (De Vlaminck et al. 2012). Furthermore, deletions of part of this domain in RecA lead to a loss of SE activity, except if SE is performed at alkaline pH (Lusetti et al. 2003). It is not impossible that Sak4 promotes SE in these condition, and this remains to be tested. It may also be that an accessory factor remains to be discovered. For example, sak4 genes often co-occur with a nearby helicase gene on phage genomes (Supplementary Table 1 in Chapter III).

Another question must be asked. RecA is sensitive to the MMR system (Tham et al. 2013) and deletion of this system increases by 1000 to 10000-fold the efficiency of recombination during conjugation between E. coli and Salmonella typhimurium [START_REF] Rayssiguier | Interspecific recombination between Escherichia coli and Salmonella typhimurium occurs by the RecABCD pathway[END_REF][START_REF] Stambuk | Mechanism and control of interspecies recombination in Escherichia coli. I. Mismatch repair, methylation, recombination and replication functions[END_REF]. So if RecA is regulated by the MMR system, is Sak4 as well? Or does Sak4, similarly to Redβ, escape the MMR control, as there are too many recombination events during phage infections?

2) … compared with other core-only RecA

In Eukaryotes, core-only RecA form complexes with other Rad51-like proteins having a Nt domain.

This is for instance the case in human, where Xrcc2 belong to the BCDX2 complex (Yokoyama et al. 2004;[START_REF] Chun | Rad51 paralog complexes BCDX2 and CX3 act at different stages in the BRCA1-BRCA2-dependent homologous recombination pathway[END_REF]. This complex promotes SSA in vitro, as GST-Sak4 (Chapter III), but also permits to load Rad51 on ssDNA and to stabilize its filament, contrary to GST-Sak4 which inhibits partially RecA SE activity in vitro (Chapter III). In yeast, Csm2 and Psy3 belong to the SHU-Chapter IV: Discussion, Origin and distribution of recombinases, The ubiquitous RecA 134 complex and, as BCDX2, permit to load and stabilize Rad51 on the ssDNA [START_REF] She | Structural and SAXS analysis of the budding yeast SHU-complex proteins[END_REF]. At present, no evidence of comparable phenotypes between phage core-only Sak4 and Eukaryotic coreonly Rad51 are available.

In Archaea, apart from RadA itself, the core-only RadB stabilizes the RadA filament (Komori et al. 2000;[START_REF] Graham | An archaeal RadA paralog influences presynaptic filament formation[END_REF]. Interestingly, RadB does not degrade ATP like Sak4 (Komori et al. 2000). The other archaeal core-only aRadC is very ancestral and homologous to KaiC, a double tandem core-only RecA found in bacteria (see Chapter I, [START_REF] Liang | Knockouts of RecA-like proteins RadC1 and RadC2 have distinct responses to DNA damage agents in Sulfolobus islandicus[END_REF])).

III) Origin and distribution of recombinases

In this last part, I want to discuss the evolution and distribution of recombinases among the three (or four with viruses) kingdoms of life. [START_REF] Lin | Origins and evolution of the recA/RAD51 gene family: evidence for ancient gene duplication and endosymbiotic gene transfer[END_REF], the "Radα" which clusters both archaeal RadA and eukaryotic Rad51 proteins, that are full size and contain a similar Nt domain. More distantly related are the "Radβ" group proteins, many of them all core-only RecA (Xrcc2, RadB). Even more distant to the Radα are a group of recombination proteins found in Archeae, aRadC, which share homology to the circadian protein KaiC of cyanobacteria. We propose that the aRadC/KaiC core-only is ancestral (root of the tree), and ask whether Sak4, being a core-only RecA of phages, would be placed in such trees at the root, together with aRadC/KaiC, or rather next to RecA, in a position similar to RadB relative to RadA.

Chapter IV: Discussion, General conclusion 136 and RPA (Replication Protein A, the SSB protein in Eukaryotes) binding domain [START_REF] Park | Physical interaction between human RAD52 and RPA is required for homologous recombination in mammalian cells[END_REF][START_REF] Shen | Specific interactions between the human RAD51 and RAD52 proteins[END_REF]Ploquin et al. 2008), ( 2) Rad59 (named Rad52B in human), homologous to phage Rad52 [START_REF] Bai | A Rad52 homolog is required for RAD51-independent mitotic recombination in Saccharomyces cerevisiae[END_REF]Mott, Symington 2011), and (3) Mgm101 [START_REF] Mbantenkhu | Mgm101 is a Rad52-related protein required for mitochondrial DNA recombination[END_REF]. This last protein acts into the mitochondria to repair DNA breaks. It is more related to phage Rad52 than to Eukaryotic ones on a structural point of view [START_REF] Mbantenkhu | Mgm101 is a Rad52-related protein required for mitochondrial DNA recombination[END_REF]. However, it is encoded in the nucleus. Two evolution histories are possible: (1) The α-Protobacteria that became mitochondria possessed a prophage encoding a Rad52, which was captured by the nucleus of the Eukaryote ancestor. This gene was then duplicated to obtain the three Eukaryotic Rad52. (2) Rad52 and Rad59 come from a Rad52-containing virus (currently disappeared or undiscovered) that was captured by an Eukaryote ancestor, and Mgm101 comes from a prophage trapped in the mitochondria.

3) The virus-only Gp2.5

Gp2.5-like recombinases of phages (Lopes et al. 2010) share homology with ICP8 from eukaryotic viruses [START_REF] Kazlauskas | Two distinct SSB protein families in nucleo-cytoplasmic large DNA viruses[END_REF]. No homologs were found in Archeal viruses yet, but it may be due to the difficulty of detecting them, as this super-family constitutes the most diverse super-family. This protein is found only in viruses and we have until now little knowledge on how it works. Other living organisms had probably rare occasions to capture these genes, or this protein could be deleterious for genome stability.

IV) General conclusion

In this thesis, I have contributed to show that relaxed fidelity is a general trait in Rad52-like and Sak4like recombinases. The present discussion has tried to draw all consequences from this fact. At present, I propose that core-only RecA might be a very ancestral form of the Rad51-like super family of recombinases, that became more specialized thanks to the addition of domains. In the same way, Pour vérifier cette hypothèse, on devrait être capable de retrouver des traces de recombinaisons entre séquences divergées dans les génomes de phage par comparaison des génomes. En théorie (Figure 3), deux phages ancestraux ayant une identité moyenne de 60% sur tout leur génome avec deux zones de plus forte homologie, à environ 80% d'identité, peuvent s'échanger les gènes compris entre ces deux zones moins divergentes grâce à leurs recombinases. Plus tard, lorsqu'on compare le nouveau phage, mosaïque des deux phages ancestraux, à l'un des parents, on devrait observer une forte zone d'homologie (appelée mosaïque pour plus de simplicité), qui correspond à la zone récemment échangée, entourée de zones de moins forte homologie, mais supérieure à celle du reste de l'alignement des deux génomes, qui correspondrait aux traces de Il est difficile de conclure sur l'activité de Sak4 en général, puisque nous avons caractérisé celle de HK620 seulement in vivo et celle de PA73 seulement in vitro. Cependant, ces différentes expériences nous on permis de constater qu'à l'instar de toute les recombinases de phages, Sak4 fait de l'appariement simple brin. Pour RecA, cette activité bien qu'observée in vitro, est inhibée in vivo.

Nous avons aussi vu qu'in vivo, la SSB accompagnant Sak4 sur HK620 est suffisante pour que Sak4 puisse faire de l'appariement simple brin, alors qu'in vitro pour la Sak4 de PA73, la SSB ne stimule en rien l'appariement simple brin, qui est efficace, même pour la protéine fusion GST-Sak4.

Chapitre IV : Discussion

Je replacerai ici mon travail de thèse dans le cadre de différents travaux qui ont tenté de tracer l' histoire évolutive et les activités de chaque famille de recombinases. Je reviendrai également sur les effets que pourraient avoir ces recombinases sur la structure des génomes, à la fois des bactéries et des phages.

Résumé

La diversité des génomes de virus infectant les bactéries, les bactériophages (ou phage en abrégé), est telle qu'il est difficile de les classer de manière satisfaisante, la notion d'espèce elle-même ne faisant pas accord dans la communauté scientifique. A la racine de cette diversité, un des facteurs clé est la recombinaison de l'ADN, qui est élevée chez les bactériophages, et permet des échanges de gènes entre entités parfois fort différentes. Mes travaux se sont centrés sur la recombinaison homologue chez les bactériophages, et en particulier sur la protéine centrale de ce processus, la recombinase. J'ai montré pour deux grands types de recombinases phagiques, de type Rad52 et Sak4, que celles-ci étaient beaucoup moins fidèles dans le processus de recombinaison, comparées à la recombinase bactérienne RecA. De plus, pour Sak4, j'ai observé que cette recombinaison se produisait par appariement simple brin, et qu'elle dépendait entièrement in vivo d'une SSB phagique, dont le gène est situé à proximité du gène sak4 sur le chromosome du phage. Les échanges génétiques sont donc grandement facilités pour les phages contenant ce type de recombinases, mais ils ne sont pas non plus anarchiques : la recombinaison s'observe jusque 22% de divergence, mais deux séquences à 50% de divergence ne peuvent recombiner. Tout se passe donc comme si la notion d'espèce devait être élargie chez les phages par rapport aux bactéries, pour inclure dans un même groupe des génomes portant des traces d'échanges récents de matériel génétique par recombinaison homologue (ce que l'on appelle le mosaïcisme).

Summary

The diversity of the viruses infecting bacteria (bacteriophages, or phages for short) is so important that it is difficult to classify them in a pertinent way, and the species notion itself is a matter of debate among specialists. At the root of this diversity, one of the key factors is DNA recombination, which occurs at high levels among phages, and permits gene exchanges among entities that are sometimes very distant. My research has focused on homologous recombination in phages, and in particular on the protein that is key to the process, the recombinase. I have shown, for two different types of recombinases, Rad52-like and Sak4-like, that their fidelity was relaxed, compared to the bacterial recombinase, RecA. Moreover, for Sak4, a protein that had not been studied before, I showed that recombination occurs by single strand annealing, and that it is strictly dependent in vivo on the co-expression of its cognate SSB protein, whose gene is often encoded nearby in phage genomes encoding sak4. Genetic exchanges are therefore greatly facilitated for phages encoding these types of recombinases. Nevertheless, exchanges are not anarchical: recombination is seen up to 22% diverged substrates, but 50% diverged DNA sequences will not recombine. It may be that the species notion should be enlarged for phages, so as to include into a same group all phages exhibiting traces of recent exchanges of genetic material (the so-called mosaicism).

  Introduction .............................................................................................................................................. ................................................................................................................................................ ....................................................................................................................................... ........................................... ................................................................. .................................................................................... .................................................... .................. ...... .................. ........................................................................................................................................ .............................................................................................................................................. ........................................................................................................................................... ........................................................................................... .......................................................................................................................................... ..................................................................................................................... ............................................................................................................ .................................................... ............................................... ............

  Sak4 proteins are hardly soluble The phage-encoded SSB-like proteins have affinity for ssDNA GST-Sak4 PA73 does not degrade ATP GST-Sak4 PA73 does not promote strand exchange, nor stimulates the RecA reaction GST-Sak4 PA73 performs SSA in vitro, and anneals up to 20% diverged sigle styranded DNAs Discussion Sak4 has SSA activity The in vitro SSA activity of RecA E.coli is inhibited in vivo Redβ λ and GST-Sak4 PA73 tolerate the presence of mismatches during SSA References Supplementary Text, Tables and figures Chapter IV: Discussion ........................................................................................................................ I) Mosaic acquisition: causes and consequences ...................................................................... 125 1) Phage recombinases, a major cause of mosaïcism ............................................................ 2) Consequences on genomes ................................................................................................. 130 3) A new definition of phage species? ..................................................................................... 132 II) The phage core-only RecA Sak4… ............................................................................................ 133 1) … compared with RecA ........................................................................................................ 133 2) … compared with other core-only RecA .............................................................................. 133 III) Origin and distribution of recombinases ................................................................................. 134 1) The ubiquitous RecA ............................................................................................................ 134 2) The phage Rad52 domesticated by Eukaryotes .................................................................. 135 3) The virus-only Gp2.5 ............................................................................................................ 136 IV) General conclusion .................................................................................................................. 136 References ........................................................................................................................................... 137 ANNEXES .............................................................................................................................................. 149 ...................................................................................................................... ................................................................................................................................................ ........................................................................................................................................ ....................................................................................................................................... ....................................... ............................................................... ...................................... .................................................................................................... ...................................................... ................................................................................................ ........ ......................................................................................... ................................. ............................................................... ..................................................................................................................... ..................

Figure 1 :Figure 2 :

 12 Figure 1: Universal phylogenetic tree in rooted form, showing the three domains. ______________ Figure 2: Comparative genome content of 13 Listeria chromosomes and L. innocua plasmid pLI100.

Figure 3 :Figure 4 :Figure 5 :Figure 6 :Figure 7 :Figure 8 :Figure 9 :Figure 11 :Figure 12 :Figure 13 :Figure 15 :Figure 16 :Figure 17 :Figure 18 :

 345678911121315161718 Figure 3: Phage life cycles. __________________________________________________________ Figure 4: Genome organization of two virulent phages: RB49 and Aeh1 (T4 family). ____________ Figure 5: Alignment of the genetic maps from the lactis phages ____________________________ Figure 6: Polymerase slippage mecanism ______________________________________________ Figure 7: DSB Repair by the NHEJ Complex _____________________________________________ Figure 8: Proposed model for MMEJ. __________________________________________________ Figure 9: Structure of the three phage recombinases families. ______________________________ Figure 10: Surface cutaways showing proposed models for RecBCD inhibition by the Gam protein. _ Figure 11: Recombination Models. ____________________________________________________ Figure 12: Experimental Approach of De Vlaminch et al. __________________________________ Figure 13: Regulation of RecA activity. _________________________________________________ Figure 14: A model of the evolutionary history of recA/RAD51 gene family. ___________________ Figure 15: Model of Redβ quaternary structure. _________________________________________ Figure 16: Model for recombination at the replication fork. ________________________________ Figure 17: Artistic renderings depicting the models for two proposed mechanisms for annealing. __ Figure 18: Presynapsis pathway in bacteriophage T4 homologous recombination. ______________ Chapter II:

Figure 1 :

 1 Figure 1: Recombinats between λ and defective prophages are formed during lytic cycle.

Figure 2 :

 2 Figure 2: Position of recombination events in homology region between λ and defective prophages.

Figure 3 : 55 Figure 4 :

 3554 Figure 3: Recombinants between λ and defective prophages are formed by the Red-pathway. 55 Figure 4: Formation of Φ80 hybrids also depend on phage recombinases.

Figure 5 :

 5 Figure 5: High efficiency of recombination of two other Rad52 recombinases.

Figure 6 :

 6 Figure 6: Heat map of mosaic characteritics in temperate and defective phages.

Figure 7 :Figure 1 :

 71 Figure 7: Rationale for the bio-informatics detection of mosaics. Supplémentary Figures.Chapter III:Figure 1: Genetic Organization of homologous recombination partners in four phages.

Figure 2 :

 2 Figure 2: Recombineering activity.

Figure 3 :

 3 Figure 3: Purified proteins on gel.

Figure 4 :Figure 5 :

 45 Figure 4: Gel shift of ssDNA by SSB proteins.

Figure 7 :

 7 Figure 7: Annealing kinetics of GST-Sak4 PA73 .

Figure 8 :Figure 19 :Figure 20 :Figure 21 :

 8192021 Figure 8: In vitro SSA activity of recombinases. Supplémentary Figures. Chapter IV: Figure 19: Design of the strand exchange reaction promoted by Redβ. ______________________ Figure 20: Representation of the pnuC:hcr junction observed by Vine et al. ___________________Figure 21: Where to place Sak4 in the RecA-like tree? ___________________________________

Figure 1 :

 1 Figure 1 : Universal phylogenetic tree in rooted form, showing the three domains. Branching order and branch lengths are based upon rRNA sequence comparisons.The position of the root was determined by comparing sequences of pairs of paralogous genes that diverged from each other before the three primary lineages emerged from their common ancestral condition. The numbers on the branch tips correspond to the following groups of organisms. Bacteria: 1, the Thermotogales; 2, the flavobacteria and relatives; 3, the cyanobacteria; 4, the purple bacteria; 5, the Gram-positive bacteria; and 6, the green nonsulfur bacteria. Archae: the kingdom Crenarchaeota: 7, the genus Pyrodictium; and 8, the genus Thermoproteus; and the kingdom Euryarchaeota: 9, the Thermococcales; 10, the Methanococcales; 11, the Methanobacteriales; 12, the Methanomicrobiales; and 13, the extreme halophiles. Eucarya: 14, the animals; 15, the ciliates; 16, the green plants; 17, the fungi; 18, the flagellates; and 19, the microsporidia.[START_REF] Woese | Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya[END_REF]

Figure 2 :

 2 Figure 2: Comparative genome content of 13 Listeria chromosomes and L. innocua plasmid pLI100. The outermost circle indicates the source of each gene in the pan-genome with each gene represented by a constant width wedge. Starting at the top of the figure (0 Mb) and movingclockwise, all EGD-e genes are arranged in chromosomal order. Continuing clockwise, all genes not present in EGD-e are grouped by strain (as indicated by segment labels). Genes in the F2365 segment are present in F2365, but absent from EGD-e, and genes in the Clip81459 segment are present in Clip81459, but absent from F2365 and EGD-e, and so on. In this way, each gene is represented only once in the diagram. Gene order in all segments except EGD-e is monotonically increasing, but discontinuous, since shared genes may be represented in other segments. Internal circles indicate gene presence (solid color) or absence (unfilled) of each gene in each of the 13 strains examined. Circles from outer to inner are in the same order as strains on the outer circle, starting with EGD-e, followed by F2365, etc. L. monocytogenes strains are in blue; L. marthii is in green; L. innocua strains are in gold; L. welshimeri is in orange; L. seeligeri strains are in red; L. ivanovii subsp. londoniensis is in purple. The location, in the EGD-e genome, of the prfA virulence cluster, conjugative transposon tn916 and prophage A118 are specifically indicated.[START_REF] Den Bakker | Comparative genomics of the bacterial genus Listeria: Genome evolution is characterized by limited gene acquisition and limited gene loss[END_REF]

Figure 3 :

 3 Figure 3: Phage life cycles. When a phage binds its receptor on a bacterium, it injects its genome.Virulent phages enter in the lytic cycle: the host genome is sometimes degraded (as represented here), and the phage multiplies itself. Capsid proteins are expressed and assembled. The phage genetic material is then encapsidated and the lysis of the cell releases virions. Temperate phages have another option after genome injection. They can enter into the lysogenic cycle instead of the lytic cycle in certain conditions. The phage genome is integrated into the bacterial genome and replicated at the same time as the bacterial chromosome. The phage is then said to be in a prophage state. After a stress, the prophage is induced and the phage enters in the lytic cycle.

Figure 4 :

 4 Figure 4: Genome organization of two virulent phages: RB49 and Aeh1 (T4 family).Genome maps of the phages are drawn to scale and, in each, the outer rings show the scales (in kb), the middle rings (yellow arrows) the T4 homologues, and the inner rings (red arrows) the ORFs with no homology to T4. T4 genes that have been rearranged (radiating blue bars) are indicated in their new locations. Various T4 genes are given as landmarks on the outside of the rings and the hyperplastic regions are indicated. The virion modules, tail fiber genes, replication modules and tRNA cluster are indicated by gray boxes.[START_REF] Comeau | Modular architecture of the T4 phage superfamily: a conserved core genome and a plastic periphery[END_REF]

Figure 5 :

 5 Figure 5: Alignment of the genetic maps from the L. lactis prophage bIL309 with the L. lactis prophage bIL286 and temperate L. lactis phage BK5-T.The open reading frames are color coded according to their predicted function (Green, predicted DNA packaging and head morphogenesis genes; brown, predicted head-to-tail joining genes; dark blue, tail morphogenesis genes; light blue, tail fiber genes; mauve, lysis genes; black, predicted lysogeny conversion genes; red, lysogeny genes; orange, DNA replication genes; yellow, transcription regulation genes ). Selected genes or genomic features are denoted. Genes encoding proteins that showed significant amino acid sequence are linked by red shading, and the percentage of amino acid identity is indicated. The degree of amino acid identity (>90, >80, >70, or <70%) is reflected in the color intensity of the red shading. Inspired from[START_REF] Proux | The Dilemma of Phage Taxonomy Illustrated by Comparative Genomics of Sfi21-Like Siphoviridae in Lactic Acid Bacteria[END_REF].

Figure 6 :

 6 Figure 6: Polymerase slippage mecanism A. (Viguera, Canceill, Ehrlich 2001) Schematic representation of the slippage process at replication fork. During DNA synthesis of a repeated sequence (step 1), the polymerase reaches a barrier on the lagging strand (a hairpin structure here) and pauses (step 2). Polymerase dissociation occurs (step 3). If the polymerase is not able to disrupt the barrier (with its strand displacement activity), then the tip of the newly synthetized strand can unpair from its template and anneal to the second repeat beyond the barrier, allowing polymerase re-loading and resumption of the synthesis (step 4). B. (Poteete 2008) Red-mediated replisome invasion/template switch. The drawing on top shows a replication fork moving through λ DNA, left to right. The drawing on bottom shows a dsDNA end processed by λ exonuclease, with the overhanging 3′ end wrapped around a Redβ ring (Passy et al. 1999) (step A). When the replisome exposes complementary bases on the strand serving as the template for lagging strand synthesis, Redβ anneals its captive strand to them (step B). Redβ positions the invading strand to serve as the new template for leading strand synthesis. The resulting joint molecule is resolved by an endonucleolytic cleavage (triangle). λ exonuclease falls off or is removed (step C). The detached lagging strand arm and unreplicated remainder of the right side of the parent chromosome is shown on top of the figure (step D). The incoming DNA now serving as the template for both leading and lagging strand synthesis, with the transferred replication fork continuing to move in the same genetic direction on its new template, is shown on bottom of the figure (step D).

Figure 7 :

 7 Figure7: DSB Repair by the NHEJ Complex The Ku complex locates to the break site (step 1), where it may serve as an end-bridging and alignment factor. Following binding to the broken DNA ends, additional processing enzymes are recruited by Ku to the break site (step 2). Ku may translocate away from the ends, allowing access by other factors to the break termini. When DNA ends are non-complementary and/or are damaged, the DNA end-processing, gap-filling, and nucleolytic activities generate DNA termini, capable of being ligated, prior to ligation (step 3). Subsequently, the broken ends are joined by an NHEJ-specific DNA ligase (step 4) and the NHEJ complex dissociates.[START_REF] Bowater | Making ends meet: repairing breaks in bacterial DNA by nonhomologous end-joining[END_REF]

Figure 8 :

 8 Figure 8: Proposed model for MMEJ. It relies on the degradation of DNA ends by the RecBCD nuclease/helicase complex (blue incomplete circle).Regions of microhomology (one to nine nucleotides, red strips) are available to promote the synapse between DNA ends. Endligation likely relies on the essential LigA. Adapted from[START_REF] Chayot | An end-joining repair mechanism in Escherichia coli[END_REF]).

Figure 9 :

 9 Figure 9: Structure of the three phage recombinases families. Model of Redβ (A) from phage λ as a representative of the Rad52-like family. Model of RecA (B) of E. coli represent the Rad51-like family. Structure of Gp2.5 (C) from the phage T7 represent the Gp2.5-like family. Adapted from (Lopes et al. 2010).

Figure 10 :

 10 Figure 10: Surface cutaways showing proposed models for RecBCD inhibition by the Gam protein. (A) Co-crystal structure of RecBCD and DNA. Models of Gam inhibition show (b) the 3′ channel occupied by helix H1 of Gam, (C) the 5′-channel occupied by helix H1 Gam and (B) the re-orientated H1 helices occupying both the 3′ and 5′ channels of RecBCD. RecB is shown in orange, RecC in blue, RecD in green.With DNA and Gam both shown in cyan. Key domains and features of the RecBCD complex are labelled. For clarity, (D) shows a different cutaway region but the same orientation.(Court et al. 2007)

  Clark and Margulies showed in 1965 that one gene is essential for recombination and repair, based on UV irradiation of E. coli K12 strain. This gene is recA (Clark, ubiquitous recombinase found in the bacterial reign. It participates to homologous recombination in vivo and repairs DNA.A) RecA activityRecA is a 38 kDa protein that catalyzes ATP hydrolysis, homologous pairing and recombination.Renaturation of complementary strand was the first activity discovered for RecA.In 1985, Bryant and

Figure 11 :

 11 Figure 11: Recombination Models. A Meselson and Weigle's break-copy recombination mechanism.The two strands of a broken chromosome fragment can form base pairs with an intact template and promote copying to the end of the template, thus producing a recombined, full-length product[START_REF] Meselson | Chromosome brekage accompanying genetic recombination in bacteriophage[END_REF]. B. Holliday's 1964 model. A pair of nonsister chromatids after meiotic DNA replication are shown; the two other chromatids, uninvolved in recombination, are not shown. A pair of same-strand nicks leads to a reciprocal exchange and formation of symmetric heteroduplex connected by a four-stranded symmetric structure now known as a Holliday junction (HJ). The HJ can be cleaved by cutting either of two pairs of strands (orientations 1 and 2). Crossovers occur when the HJ is cleaved so that only the crossing-strands connect the two homologous chromosomes. In the example shown, mismatch corrections lead to a 6:2 gene conversion[START_REF] Holliday | A mechanism for gene conversion in fungi[END_REF]. C. Charles Thomas's SSA model to obtain reciprocal recombination by annealing overlapping single strands of DNA from two chromosomes with offset nicks on both strands[START_REF] Thomas | Recombination of DNA molecules[END_REF]. D.Meselson and Radding's 1975 model. A single nicked strand is displaced by new DNA synthesis primed from the 3' end of the nick. The displaced strand can form a region of heteroduplex DNA by strand invasion and the formation of a displacement loop (D-loop). Cleavage of the D-loop leaves a single region of heteroduplex DNA adjacent to an HJ that is always distal from the initiating nick. Isomerization of the HJ and subsequent branch migration leads to the formation of a symmetric (sym. het) region of heteroduplex adjacent to an asymmetric (asym. het) segment (with only one heteroduplex region), still with the crossover point far from the initiating lesion[START_REF] Meselson | A general model for genetic recombination[END_REF]). E. The DSB repair model of[START_REF] Resnick | The repair of double-strand breaks in DNA; a model involving recombination[END_REF]. (α) A DSB is resected and one of the 3′ ends invades a template. The template strand is nicked. The paired DSB end initiates new DNA synthesis, which is displaced, allowing the original sequences to reanneal with the template. When the newly-copied strand is long enough, it can anneal with the second end, priming a second round of new synthesis and the repair of the DSB. This leads to a noncrossover repair of the DSB. (β) Crossovers can be generated by DSB repair. Here, the nicked template strand itself can anneal with the single-stranded sequences created by 5′ to 3′ resection. Strand invasion of the original DSB end creates a Holliday junction. An endonuclease nick at the base of the HJ results in connections between the molecules that result in a crossover. DNA synthesis fills in the ssDNA gaps and leads to a crossover[START_REF] Resnick | The repair of double-strand breaks in DNA; a model involving recombination[END_REF]. F. The double Holliday junction (dHJ) model of[START_REF] Szostak | The double-strand-break repair model for recombination[END_REF]. A DSB was enlarged into a double-stranded gapped region, which was subsequently resected to have 3′-ended single-stranded tails that could engage in strand invasion. The first strand invasion would produce a D-loop to which the end of the second resected end could anneal. The initial structure has one complete and one half HJ, but branch migration of the half HJ allows the formation of a second complete HJ. New DNA synthesis completes the formation of a fully ligated structure that can be resolved into crossovers if the two HJs are cleaved in different orientations. Resolution of both HJs as crossovers should leave an apparent double crossover in the middle; such outcomes are rare[START_REF] Szostak | The double-strand-break repair model for recombination[END_REF]. Adapted from[START_REF] Haber | RecA family proteins in archaea: RadA and its cousins[END_REF].

  Chapter I: Introduction, Homologous recombination, RecA 35

Figure 12 :

 12 Figure 12: Experimental Approach of De Vlaminch et al. (A) Side view of dual-molecule assay where a RecA filament that is held in the optical tweezers interacts with a coiled dsDNA that is tethered in a magnetic-tweezers configuration. Inset: schematic of the RecA filament represented with two DNAbinding sites, a primary site (1) and secondary DNA-binding site (2). (B) The four different experimental configurations I-IV used in their work. (C) Schematic outline of the setup indicating the main components. Two independently steerable optical traps are generated using a 1064nmlaser system. The beams are split and recombined using a polarizing beam splitter and focused using a high numerical aperture objective in the volume of a four-channel laminar-flow cell. Positioning and rotation of an external magnet pair allows stretching and coiling of a molecule tethered in the magnetic tweezers configuration. (D) Top view of the four-channel laminar-flow system used for the assembly of the molecular constructs. (De Vlaminck et al. 2012)

  designated as radα and radβ. In Archaea, Radα became RadA and Radβ became RadB. The ancestor of Eukaryotes acquired a bacterial copy of RecA by endosymbiosis of an α-Proteobacteria, the mitochondria. Then Radα duplicated into Rad51 and DMC1, its paralog in meiosis, and Radβ into Chapter I: Introduction, Homologous recombination, Phage recombinases 40 different paralogs named differently in Mammals and Fungi. Plants conserved the unduplicated, bacterial RecA copy, contrary to Mammals and Fungi, and acquired a second one from the Cyanobacteria endosymbiont, the chloroplast.

Figure 14 :

 14 Figure14:A model of the evolutionary history of recA/RAD51 gene family. The gene duplication that occurred before the divergence of archaea and eukaryotes gave rise to two lineages, RADα and RADβ, and, in eubacteria, recA has remained as a single-copy gene. In eukaryotes, both RADα and RADβ genes experienced several duplication events, but, in archaea, they remained as single-copy genes. Eukaryotic recA genes originated from proteobacteria (recAmt) and cyanobacteria (recAcp) recA genes after two separate endosymbiotic events. recAmts were subsequently lost in the ancestors of animals and fungi[START_REF] Lin | Origins and evolution of the recA/RAD51 gene family: evidence for ancient gene duplication and endosymbiotic gene transfer[END_REF]).

  . Erler et al. proposed an alignment in 2009 of Redβ with the eukaryotic Rad52 protein, and observed that the ring structure of Redβ without DNA had only 11 monomers and was not closed. The authors hypothesized this ring is a right helix. In the presence of ssDNA, they observed Redβ is not organized, contrary to previous observations. As previously, in the presence of dsDNA, Redβ formed a helix filament (Figure 15B, (Erler et al. 2009)). In 2010, our group also found distant homology between Rad52 and various phage recombinase: Erf, Redβ/RecT and Sak. Last year, Matsubara et al. realigned different Redβ, Rad52 and Sak recombinases (Matsubara et al. 2013). They experimentally distinguished amino acids involved in the quaternary structure (K148 and R149 in Redβ), in ssDNA binding (V77, G78, V79, W82, R152 and probably R161) and in SSA activity (K69, G78 and K132) How exactly functions Redβ in vivo is not completely understood. Two groups have shown that recombination is replication dependent. Poteete first reported in 2008 in vivo experiments with a non-replicating λ and a plasmid with conditional replication. The recombination was inhibited when the plasmid replication was prevented. He showed also that SSA was more efficient when the ssDNA to anneal was complementary to the plasmid lagging strand, compared to the leading strand. His model proposed that Redβ anneals a λ 3'-overhang ssDNA onto the lagging strand template and that the replication machinery of the plasmid switches on to the invading DNA (presented in Figure 6B (Poteete 2008)). In 2011, Maresca et al. also observed replication-dependent recombination, but

Figure 15 :

 15 Figure 15: Model of Redβ quaternary structure. (A) Models for the complex of the large Redβ ring and ssDNA (a) and the Redβ helix formed on dsDNA (b). The ssDNA is believed to wrap around the outside of the 18-subunit rings. The exposure of the bases would provide the catalytic role played by these rings in DNA annealing. The helical filaments are formed by Redβ protein on preformed dsDNA, or on dsDNA products of the Redβ-catalyzed annealing reaction. The Redβ filament induces a left-handed supercoil in the DNA, with about one supercoil turn for every 100 bp. (Passy et al. 1999) (B) Models of the Redβ quaternary structure in the absence of DNA, the disordered Redβ-ssDNA complex, and the Redβ-dsDNA complex after annealing. (a) Model of the right-handed helix of a Redβ polymer in the absence of DNA. (b) Model of the heterogeneous and disordered binding of Redβ to ssDNA and annealing initiation. (c) Model of the stable helical Redβ-dsDNA annealing intermediate, assuming that the bound DNA is stretched by a factor of 1.53 and takes a right-handed superhelical path around the left-handed Redβ helix. The two DNA strands do not cross each other with respect to the Redβ protein surface.(Erler et al. 2009)

Figure 16 :

 16 Figure 16: Model for recombination at the replication fork. Annealing of an ssDNA molecule to complementary regions on the lagging strand template at the replication fork is depicted. The ssDNA molecule comprises two flanking homology arms (~50 nt; yellow), interspaced by a heterologous sequence (light green). The Redβ annealing intermediate is shown as a curved line of red dots. The leading strand is shown in blue, lagging strand in black, DnaB helicase in light orange, the two Pol III holoenzymes are green, which are tethered to the γ/τ clamp loader (light blue), and the β sliding clamps are dark blue rings. (A) The Redβ-ssDNA protein complex anneals the 3' end first, which then primes DNA synthesis for an Okazaki fragment. (B) After replication fork progression, the second homology region becomes exposed and annealing of the 5' homology arm creates the ssDNA heteroduplex intermediate.(Maresca et al. 2010)

Figure 17 :

 17 Figure 17: Artistic renderings depicting the models for two proposed mechanisms for annealing. (a) ssDNA molecules are first bound by ICP8, and then annealing follows catalyzed by the docking and sliding of these two rings. (b) It shows the ssDNA entering a pre-formed oligomeric ICP8 ring.[START_REF] Tolun | Details of ssDNA annealing revealed by an HSV-1 ICP8-ssDNA binary complex[END_REF]

  Maher and Morrical studied the DNA binding activity of UvsX. They observed the dissociation constants of UvsX for ssDNA and dsDNA in the presence or absence of ATP or ATPγS (a nonhydrolysable form of ATP). It seems that without ATP, UvsX has a low affinity for dsDNA, and cannot bind the ssDNA at all. It has a better affinity for the dsDNA in presence of ATPγS than in presence of ATP, which means UvsX probably releases dsDNA after ATP hydrolysis. The affinity for the ssDNA is the same in presence of ATP and ATPγS. The affinity is the highest when UvsX is mixed with dsDNA

Figure 18 :

 18 Figure 18: Presynapsis pathway in bacteriophage T4 homologous recombination. (A) A dsDNA end may be nucleolytically resected to expose a 3' ssDNA tail. The Gp46 and Gp47 proteins are thought to be the major enzymes involved in the resection step. (B) The exposed ssDNA is sequestered by the Gp32 ssDNA-binding protein, which denatures secondary structure in ssDNA and keeps it in an extended conformation.(C) The UvsY recombination mediator protein forms a tripartite complex with Gp32 and ssDNA and "primes" the complex for recruitment of UvsX recombinase. (D) UvsY recruits ATP-bound UvsX protein and nucleates presynaptic filament formation. Gp32 is displaced in the process.(Liu et al. 2011)

Figure 1 .

 1 Figure 1. Recombinants between l and defective prophages are formed during lytic cycle. A, B and C: Maps of the three regions of similarity between l and MG1655 used in this study. Antibiotic resistance genes (white arrows) were inserted in the defective prophages, in the middle of the identity regions. KanR stands for the gene conferring resistance to kanamycine, the cat gene confers resistance to chloramphenicol. Identity region between l and Dlp12 (A) spans across essential lysis genes (R and Rz) and terminase genes (A and nu1), separated by the cos site. Identity regions between l and both Qin (B) and Rac (C) span across side tail fibers genes (stf and tfa). The ble gene, that confers resistance to phleomycine, was inserted between tfa and ea47, under the constitutive promoter PsacB. Blue arrows in C indicate the position of the 3 oligonucleotides used to sequence the recombinants. D: Schematic representation of the recombination assay: (1) l phage is multiplied on a strain in which an antibiotic cassette has been inserted in a region of homology. (2) The phage produced is used to lysogenize a new strain. The total number of lysogenized bacteria is determined by their phleomycine resistance, while bacteria lyzogenised by a recombinant phage are also resistant to either chloramphenicol or kanamycin. E: Frequency of l recombinants with Dlp12, Qin and Rac. Bp numbers indicate the size of the homology regions. Mean 6 standard deviation of at least 3 independent recombination assays is indicated. doi:10.1371/journal.pgen.1004181.g001

Figure 2 .

 2 Figure 2. Position of recombination events in homology regions between l and defective prophages. A: Positions of recombination events between Rac and l for 12 sequenced clones. Mismatches in similarity regions between rac and l are represented by vertical black bars. Intervals between two mismatches in which sequencing revealed that recombination occurred are colored in green, intensity representing the number of recombination events (see legend). B: Positions of recombination events between Dlp12 and l. Nine and 19 clones were sequenced on the left and right sides of cat gene, respectively. Half of the recombination events scored occurred at direct proximity of the cos site. doi:10.1371/journal.pgen.1004181.g002

Figure 3 .

 3 Figure 3. Recombinants between l and defective prophages are formed preferentially by the Red-pathway, especially when sequences are short or diverged. A: Frequency of recombinants with Dlp12, Qin and Rac, as a function of Redb and RecA presence (indicated below the bars). Both recombinases are able to catalyze the exchanges, but Redb participation is more pronounced, especially on short and diverged sequences. When both recombinases are absent, almost no recombinants are obtained. B: Role of reda, orf and rap genes

Figure 4 .

 4 Figure 4. Formation of W80 hybrids also depend on phage recombinase. A: map of the 300 bp region of 96% identity between the W80 region near attP site and E. coli gene yecD. B: Map of the 980 bp homology region between Dlp12 and W80, spanning through the essential terminase genes A and nu1. C: Highly divergent 500 bp homology region between the non-essential nin region of W80 and Dlp12. D: Frequency of recombinants for each homology region, as a function of Redb and RecA (genotypes are indicated below the bars). Mean 6 standard deviation of at least 3 independent recombination assays are indicated. doi:10.1371/journal.pgen.1004181.g004

Figure 5 .

 5 Figure 5. High efficiency of recombination of two other Rad52 recombinases.A: Principle of the assay: two 800 bp homologous sequences (oxa genes, as in[START_REF] Martinsohn | The lambda red proteins promote efficient recombination between diverged sequences: implications for bacteriophage genome mosaicism[END_REF]), inversely repeated, represented by the red and yellow rectangles, are inserted so as to flank the P L promoter in the l genome. Inverted sequences are either 100% or 78% identical. When homologous recombination occurs between the repeated sequences, the P L promoter is inverted, which leads to a phenotypic switch, because the red and gam genes are no longer expressed (see Materials and Methods). B: Recombination frequencies scored with three different pairs of phage recombinase/exonuclease, and compared to RecA pathway. The recET (from Rac prophage) and erf/ exo (from phage PA73) genes were substituted in place of the l red genes. Values shown for redab and recA pathways are those reported in[START_REF] Martinsohn | The lambda red proteins promote efficient recombination between diverged sequences: implications for bacteriophage genome mosaicism[END_REF]. Mean 6 standard deviation of at least 3 independent recombination assays are indicated. doi:10.1371/journal.pgen.1004181.g005

Figure 6 .

 6 Figure 6. Heat map of mosaic characteristics in temperate and defective phages. Density of mosaics found among pairs of phages (lower part of the diagonal, purple to green colours), and average number of traces of recombination detected at the boundary of mosaics (higher part, yellow to red colours). The density is the number of mosaics per 10 kb of phage genome. Names of l-like phages are in red. doi:10.1371/journal.pgen.1004181.g006

Figure 7 .

 7 Figure 7. Rationale for the bio-informatics detection of mosaics. Upper panel: two ancestral phages A and B recombine across two regions of partial identity (light grey squares). As a consequence, the new piece of DNA in the recombined phage C, when compared to its parent B providing the mosaic, exhibits a 100% identity region (dark grey square), flanked by the two partially identical sequences (light grey), above the background level b of low identity shared by B and C. Upon alignment of genome C with B around the mosaic (lower panel), if the regions flanking the mosaic have a percentage of identity above the background level of identity ($b+10%), they will be counted as traces of homologous recombination (HR trace). doi:10.1371/journal.pgen.1004181.g007

Figure S1

 S1 Figure S1 Distribution of perfect identity segment sizes in the different homology regions. Lengths of homology regions with l (A) and W80 (B) are shown in blue and red for the regions placed left and right from the antibiotic cassette, respectively. Green bars indicate the segments in which a recombination event was detected. (C) Purple: Length distribution of homology regions for the oxa5/oxa7 sequences (P L inversion assay). Green: segments in which a recombination event was detected. (D) Numbers of MEPS (Minimal Efficiency Pairing Segment), defined as the minimal length of strict identity to initiate pairing, for Redb and RecA in the different homology regions. (DOCX) Figure S2 Single burst growths of Url, Urlble and Urlble Dredb (lSOC27). The insertion of the phleomycin resistance gene (ble) does not modify either the burst size or the latency period of the phage. Deletion of the redb gene reduces the burst size by 5 fold, but the latency period is unchanged. The experiment has been repeated four times independently, and a representative result is shown. (DOCX) Figure S3 Examination of Rac prophage replication. As the Rac prophage possesses an active origin of replication, oriJ, we investigated by semi-quantitative PCR if l infection activates Rac replication. Cells infected or not with l at an m.o.i. of 1, were

  Figure S1 Distribution of perfect identity segment sizes in the different homology regions. Lengths of homology regions with l (A) and W80 (B) are shown in blue and red for the regions placed left and right from the antibiotic cassette, respectively. Green bars indicate the segments in which a recombination event was detected. (C) Purple: Length distribution of homology regions for the oxa5/oxa7 sequences (PL inversion assay). Green: segments in which a recombination event was detected. (D) Numbers of MEPS (Minimal Efficiency Pairing Segment), defined as the minimal length of strict identity to initiate pairing, for Redb and RecA in the different homology regions.

  FIGURE S2

Figure S3

 S3 Figure S3 Examination of Rac prophage replication. As the Rac prophage possesses an active origin of replication, oriJ,we investigated by semi-quantitative PCR if l infection activates Rac replication. Cells infected or not with l at an m.o.i. of 1, were harvested 30 min after infection for total DNA purification. DNA was purified after phenol-chloroform extraction and ethanol precipitation steps. PCR was realized on 50 pg of total DNA, with 22 cycles of amplification. 1 to 2: PCR with primers framing oriJ does not indicate Rac replication following l infection (line 2, MG+l) compared to the non-infected MG1655 strain (line 1,MG). 3 to 4: internal controlwith primerswithin the gyrB gene.5&6:PCR on the l gene Q shows l replication in the MG+ l sample (lane 6).

Figure S5 :

 S5 Figure S5: Distribution of mosaic sizes in the 4 categories of comparisons.

Figure 1 :

 1 Figure 1: Genetic organization of homologous recombination partners in four phages. P22,

Figure 2 :

 2 Figure 2: Recombineering activity. A. Assay: a recA mutS E. coli [Cm S ], carrying a plasmid coding for a recombinase under the control of an arabinose inducible promotor (1), is electroporated with an 81-mer oligonucleotide (2) complementary to a mutated cat gene (cat SS , see Suppl. Text). Integration of the oligonucleotdide at the replication fork (3), confers a Cm R phenotype to the bacterium. B. recombineering efficiency for four phage recombinases, with or without their partners described Fig.1 : RecA E.coli , Sak4 HK620 , Sak4 PA73 and Redβ λ . C. Effect of the oligonucleotide divergence relative to the target catSS gene, on the efficiency of recombineering. The recombinases were tested with all their partners. White bars show the detection limit.

Figure 3 :

 3 Figure 3: Purified proteins on gel.

Figure 4 :Figure 5 :

 45 Figure 4: Gel shift of ssDNA by SSB proteins. 10nM of GO47 was mixed with SSB proteins for 15minutes and then loaded on a 10% acrylamide/bis-acrylamide (19:1) gel. SSB E.coli was used at 0.25µM, 0.5µM, 1µM and 2µM. SSB HK620 and SSB PA73 were used at 0.5µm, 1µM, 2µM, 4µM and 8µM.

Figure 6 :

 6 Figure 6: GST-Sak4 PA73 does not promote strand exchange. A. Scheme of the strand exchange reaction: circular ssDNA (ssc) bound to the recombinase reacts with its homologous linear dsDNA (dsl) to form joint molecules (jm) between ssc and dsl. The final products of the strand exchange are a nicked circular dsDNA (nc) and linear ssDNA (ssl). B. Different order of addition of proteins (1µM of RecA E.coli , 2µM of GST-Sak4 PA73 and 0.5µM of SSB E.coli ) before addition of the dsl. The order of addition of RecA E.coli and GST-Sak4 PA73 is specified by 1 and 2, or added at the same time. SSB E.coli is always added last (see methods). C. Same experiment with 2µM of RecA E.coli . D. Same as C but 1µM of SSB PA73 was used instead of SSB E.coli .

Figure 7 :

 7 Figure 7: Annealing kinetics of GST-Sak4 PA73 . Annealing kinetic without ATP, the same curves are obtained with 1 mM ATP. The error bars represent the lower and higher values obtained.

Figure 8 :

 8 Figure 8: in vitro SSA activity of recombinases. A and C. Representative gels of SSA assay without (A) or with (C) 1mM ATP respectively: the GST control, RecA E.coli and Sak4 PA73 -GST. The lower band represents the Cy5-tagged GO47. The higher band is the dsDNA corresponding to the annealing of GO47 and the more or less diverged complementary oligonucleotide (described in Table2). B and D. Quantification of SSA activity without (B) or with (D) 1mM ATP respectively for the GST control, RecA E.coli , Redβ λ , UvsX T4 and Sak4 PA73 -GST in function of divergence (3 measures at each point, see supplementary Figure 4 for details).

3 :

 3 SSA of all recombinases used in this study, and of GST, in the absence of ATP. A representative gel is shown for each recombinase on the left of the figure (time point 7 min). On the right part, the mean (3 replicates) percents of annealing were quantified for each reaction. The error bars represent the minimum and maximum annealing measured.

1 )

 1 acquisition: causes and consequencesAs explained in the introduction, temperate phage genomes are mosaic. Here I will discuss the contribution of homologous recombination to this mosaïcism as well as the consequences for both host and phage genomes. I will also address the question of phage classification and the notion of species under the homologous recombination point of view. Phage recombinases, a major cause of mosaïcism Phage recombinases seem to play an important role in mosaïcism. It was proposed that their relaxed fidelity during recombination might participate to mosaic acquisition in Enterobacteria phages, which

Figure 19 :

 19 Figure 19: Design of the strand exchange reaction promoted by Redβ.A labelled 43-mer oligonucleotide that had been thermally annealed to single strand circular M13 DNA to form partial duplex was displaced in the presence of Redβ. By an overlapping 63-mer, called the donnor strand, that had 20 additional nucleotide residues entirely at its 3' or 5' end, relative to the 43-mer.[START_REF] Li | The beta protein of phage lambda promotes strand exchange[END_REF]

Figure 20 :

 20 Figure 20: Representation of the pnuC:hcr junction in the strain cinstructed by Vine et al. (Vine et al. 2010). The pnuC and hcr sequences were aligned. Black bars represent the homologous bases. The final sequence after recombination is represented in blue.

  Chapter IV: Discussion, Mosaic acquisition: causes and consequences, A new definition of phage species? 132 Rad52-like recombinases, without letting them directing the recombination process. Rad52 serves as a mediator in yeast between the SSB protein RPA and the recombinase Rad51 ((Krogh, Symington 2004) for review).

Figure 21 :

 21 Figure 21:Where to place Sak4 in the RecA-like tree? A scheme summarizing various phylogenetic studies on RecA-like proteins is shown. On the right part, the bacterial RecA. On the left part, following Lin et al. nomenclature[START_REF] Lin | Origins and evolution of the recA/RAD51 gene family: evidence for ancient gene duplication and endosymbiotic gene transfer[END_REF], the "Radα" which clusters both archaeal RadA and eukaryotic Rad51 proteins, that are full size and contain a similar Nt domain. More distantly related are the "Radβ" group proteins, many of them all core-only RecA (Xrcc2, RadB). Even more distant to the Radα are a group of recombination proteins found in Archeae, aRadC, which share homology to the circadian protein KaiC of cyanobacteria. We propose that the aRadC/KaiC core-only is ancestral (root of the tree), and ask whether Sak4, being a core-only RecA of phages, would be placed in such trees at the root, together with aRadC/KaiC, or rather next to RecA, in a position similar to RadB relative to RadA.

  Rad52-like recombinases, that are unfaithful, were domesticated by Eukaryotes by the acquisition of a supplementary domain, like in Eukaryotic Rad52, or by being used as cofactor of recombination, like Rad59. Finally, I propose a new method of classification of phages, excluding defective prophages, based on the relaxed fidelity of the phage recombinases: two phages capable of exchanging genetic material would belong to the same species. 149 ANNEXES mesurer précisément la contribution de différentes recombinases phagiques et de RecA aux échanges de matériel génétique, en fonction de la divergence des séquences recombinantes. Ce travail est présenté dans le chapitre II. Dans un deuxième temps, j'ai décidé de me tourner vers l'étude de Sak4, encore très peu étudiée. J'ai utilisé des approches de génétique et de biochimie pour caractériser cette protéine. La question principale a été de savoir si son activité était à l'image de RecA ou plus proche des Rad52. Ce travail est présenté au chapitre III Chapitre II : Les phages tempérés acquièrent de l'ADN provenant des prophages défectifs par un mécanisme de recombinaison homologue relâchée : le rôle des recombinases de la famille des Rad52. Les phages sont très divers. Des analyses de comparaison génomique suggèrent qu'ils ont un fort taux de transfert horizontal, surtout les phages tempérés à ADN double brin. Le mécanisme moléculaire sous-jacent est encore inconnu et sujet à plusieurs hypothèses. Notre hypothèse part de l'observation qu'une recombinase de la famille des Rad52, Redβ, encodée par le phage λ, a une fidélité diminuée par rapport à la recombinase de l'hôte, RecA. Nous proposons que les recombinases de phages,à cause de cette fidélité réduite sont capables de promouvoir le transfert de gènes entre phages même si les séquences sont éloignées. Les phages tempérés étant capables de s'insérer dans le chromosome de leur hôte, la probabilité de rencontre entre le phage infectant une bactérie et divers prophages déjà présents dans le chromosome bactérien, même défectifs, est augmentée, au-delà des échanges entre deux phages différents infectant simultanément la même bactérie. Enfin, nous nous sommes aussi demandés si les phages utilisaient uniquement leur propre recombinase ou s'ils étaient capables d'utiliser à la fois la leur et celle de l'hôte. Dans un premier temps, nous avons cherché à comprendre quelles recombinases participaient à un événement de recombinaison entre le phage λ et divers prophages défectifs d'E. coli K12. Pour cela, nous avons identifié 3 régions homologues entre prophages défectifs et le phage λ : dans Dlp12, une région de 3500 pb autour du site cos présente 98% d'identité avec λ ; dans Qin, une région de 700 pb présente 95% d'identité avec le locus tfa de λ ; et enfin dans Rac une région de 700 pb présente 85% d'identité avec le locus stf de λ. Au centre de chacune de ces zones, un gène de résistance à un antibiotique (kanamycine ou chloramphénicol) a été introduit de telle manière à ce que λ puisse capturer ce gène par double événement de recombinaison homologue. Le principe est simple : un dérivé de λ possédant un marqueur de résistance à la phléomycine (λ P ), délété ou non de son gène de recombinason homologue redβ, infecte une des souches d'E. coli ayant un prophage modifié comme décrit plus haut. Cette souche est de plus délétée ou non de son gène de recombinaison homologue recA. Lors de l'infection, λ P se multiplie par cycles lytiques, et a la possibilité de capturer le gène de résistance placé dans le prophage. Pour dénombrer les phages recombinants, le surnageant contenant les phages après la lyse des bactéries est récupéré, puis les phages sont lysogénisés dans une souche sensible aux antibiotiques. Les phages totaux sont comptabilisés par sélection sur phléomycine, et les phages recombinants par sélection sur chloramphénicol ou kanamycine, selon le marqueur placé dans le prophage. Le ratio de ces deux dénombrements permet de comptabiliser les fréquences de recombinaison lors du cycle lytique. Aucun recombinant n'a été obtenu en l'absence des deux recombinases, Redβ et RecA, alors que des fréquences de recombinaison allant jusqu'à 10 -4 sont observées en présence des recombinases (région de 3500 pb d'homologie). Ce résultat souligne l'essentialité des recombinases dans le processus de transfert horizontal entre phages et prophages et, par extension, entre phages. Un effet très net de la taille de la région homologue est observé : entre 700 pb et 3500pb, la fréquence augmente d'un facteur 100 (les deux régions sont à 98% identiques entre elles). Il est connu que la fréquence de recombinaison homologue augmente proportionnellement à la taille des séquences d'homologie. Il est également connu que la présence du site cos, un site de coupure reconnu par la terminase du phage, augmente la probabilité d'avoir des événements de recombinaison à ce locus précis. Sur des séquences longues à forte identité, RecA et Redβ semblent participer à parts égales aux événements de recombinaison : chaque simple mutant diminue d'un facteur deux environ la fréquence de recombinaison. De manière intéressante, Il n'y a pas de différence entre la fréquence de recombinaison des séquences à 95% d'identité et celles entre séquences de 85% d'identité de même longueur (700 pb).Pour ces séquences plus courtes, divergées ou non, Redβ joue un rôle majeur, et RecA un rôle mineur : elle participe environ à 1/10 ème des événements de recombinaison (Figure1).

Figure 1 :

 1 Figure 1 : Les recombinants entre λ et les prophages défectifs sont formés préférentiellement par Redβ, surtout si les séquences sont courtes. Moyenne des fréquences des recombinants avec Dlp12, Qin et Rac en fonction de la présence ou non de Redβ et RecA. Les barres d'erreur sont des écart-types calculé à partir de 3 tests de recombinaison indépendants.

Figure 2 :

 2 Figure 2 : Haute efficacité de recombinaison de deux autres Rad52. Moyenne des fréquences d'inversion du promoteur P L suivant la présence de différentes recombinases précisées sur le coté. Les barres d'erreur sont des écart-types calculés à partir de 3 tests de recombinaison indépendants.

  recombinaison homologue sur séquences divergées. Pour tester cette idée, 84 génomes de phages infectant E. coli ont été analysés, dont 24 tempérés, 26 virulents et 34 prophages défectifs. Ces génomes ont été comparés deux à deux par BLAST et les résultats ont été filtrés. Seules les régions ayant plus de 90% d'identité sur au moins 100 pb ont été conservées. Ces régions sont considérées comme des mosaïques, des zones d'échanges récents autour desquelles il serait possible de trouver des traces de recombinaison entre séquences divergées. Pour rechercher ces traces de recombinaison, les 2 kb flanquant la mosaïque sur chaque phage sont analysés : un alignement par programmation dynamique (Needlemann et Wunsch) est réalisé. Le pourcentage d'identité de cet alignement est ensuite déterminé par fenêtres successives de 50 pb. Il y a trace de recombinaison si l'on détecte au flanc de la mosaïque une des régions ayant un score d'identité intermédiaire entre la mosaïque et le reste du génome (un score d'identité inférieur d'au moins 10% à celui de la mosaïque et supérieur d'au moins 10% au score médian des identités trouvées pour toutes les fenêtres). Les

Figure 3 :

 3 Figure 3 : Schéma de la détection bioinformatique des mosaïques. 2 phages ancestraux A et B partageant des zones d'identité, où il y a possibilité de recombiner, peuvent s'échanger du matériel génétique pour donner le phage C. Le pourcentage d'identité attendu le long de l'alignement de B contre C est présenté dans le graphique placé sous le schéma, la mosaïque est à 90% d'identité et des traces de recombinaisons entre séquences divergées autour sont à 70% d'identité.

Figure 4 :

 4 Figure 4 : Activité d'appariement à la fourche de réplication. Efficacité d'appariement de 4 rececombinases avec leurs partenaires : RecA d'E.coli, Sak4 de HK620, Sak4 de PA73 et Redβ de λ.

  Les recombinases de type Rad52 sont présentes dans les génomes de phages, mais pas chez les bactéries. Chez les Eucaryotes, on retrouve cette protéine fusionnée à un domaine C-terminal, qui réoriente la fonction de la protéine vers une activité de chargement de Rad51. Je propose que cette famille de recombinases soit une famille spécifique des génomes de phages, spécialisée dans la recombinaison entre séquences divergées, qui a été domestiquée par les Eucaryotes à partir d'une copie apportée par un génome de bactérie endocytée par ces derniers, bactérie qui contenait peutêtre un prophage. Les plus étudiées des recombinases de cette famille sont la recombinase du phage λ, Redβ, et la recombinase du prophage rac d'E. coli, RecT. Ces recombinases ont plusieurs activités, dont une est plus efficace que les autres. La plus efficace de ces activités est l'appariement simple brin, qui consiste à apparier deux ADN simple brin complémentaires d'origines différentes. Le modèle le plus approprié pour expliquer nos résultats consiste à proposer qu'à partir d'un fragment linéaire d'ADN double brin, une exonucléase dégrade ce fragment de sorte qu'il soit rendu entièrement simple brin. Redβ peut alors se fixer à ce substrat, et chercher de l'homologie à la fourche de réplication, le fragment d'ADN simple brin prend alors le rôle de fragment d'Okazaki. Cependant, ce modèle ne permet pas d'expliquer les recombinaisons allant jusqu'à un échange de grands fragments, d'une taille supérieure à la région laissée simple brin avant synthèse des fragments d'Okasaki (1 à 2 kb), comme ceux qui s'échangent dans le test de capture de gène de résistance, décrit plus haut. La deuxième activité, une certaine forme d'échange de brin, pourrait mieux expliquer des échanges de grands fragments. En effet Redβ, une fois qu'elle a apparié deux Si on considère que l'activité d'appariement simple brin des recombinases de phages est liée à leur capacité à recombiner les séquences divergées, on peut considérer qu'elles sont à la fois un atout majeur pour l'évolution des génomes de phages et un désavantage pour les génomes bactériens. En effet les phages sont très divers et, lorsqu'on les compare au niveau de la séquence de leur génome en acides nucléiques, ils sont très divergés les uns par rapport aux autres, y compris au sein d'une même famille. Ils échangent cependant du matériel génétique, surement grâce à la faible fidélité et haute efficacité de leur recombinases, pour ceux qui en portent une. Quant aux génomes bactériens, il est dangereux pour eux d'avoir ce genre de recombinases. En effet les recombinases de l'hôte préservent l'intégrité du chromosome bactérien grâce à leur activité fidèle. Les recombinases qui apparaissent dans les prophages ne sont pas, ou très peu, exprimées afin d'éviter les dommages, comme par exemple des délétions majeures ou des inversions chromosomiques. Enfin, il serait peutêtre envisageable de dériver de ces travaux une notion d'espèces pour les phages. En effet, si des phages sont capables d'échanger du matériel génétique, grâce aux recombinases ou par un autre procédé, de manière à créer une progéniture viable, alors sans doute ces phages appartiennent à la même espèce. Dans cette thèse, j'ai montré que la fidélité relâchée est un trait général des recombinases des familles des Rad52 et des Sak4. La discussion ci-dessus a tenté de tracer les conséquences de ce fait. Je propose que l'ancêtre commun aux Rad51 ne soit composé que du domaine coeur commun à toutes les protéines de la superfamille, qui par la suite a acquis des domaines supplémentaires pour se spécialiser. De la même manière, les recombinases de la famille des Rad52, qui ont une fidélité relâchée, ont été domestiquées par les eucaryotes par l'acquisition de domaines supplémentaires, comme la Rad52 eucaryote, ou utilisés comme cofacteur de recombinaison, comme Rad59. Enfin, je propose une nouvelle méthode de classification des phages, en excluant les prophages défectifs, en se basant sur la fidélité diminuée des recombinases phagiques : deux phages capables d'échanger du matériel génétique appartiendraient à la même espèce.

Table 1 .

 1 Data collected on mosaics, by categories of genome pairs compared.

	Phages pairs	T-T*	T-D	D-D	V-V	V-others
	N genome pairs analysed (p)	266	807	549	289	1508
	N of Blast matches	711	895	670	16	0
	% of IS matches	9.7	26.7	33.6	0.0	0
	N of mosaics analyzed for HR traces (m)	635	631	430	16	0
	N mosaics/genome pair (m/p)	2.39	0.78	0.78	0.06	0
	Median mosaic length (bp)	404	494	458	131	0
	Median mosaic id%	96.1	94.8	95.1	91.5	0
	N of mosaics without HR traces	432	411	298	5	0
	N with 1 trace of HR	169	171	106	6	0
	N with 2 traces of HR	34	49	26	5	0
	P ($1 trace of HR at random)	,10 216	,10 216	,10 216	1.8 10 214
	*T, temperate, D, defective, V, virulent.
	doi:10.1371/journal.pgen.1004181.t001

Table S1

 S1 Bacterial and phage strains used in this study.(DOCX)

	Table S2 Differences between the sequence of l (lPapa) and the
	Url-ble phage used in this study.
	(DOCX)
	Table S3 Primers used in this study.
	(DOCX)
	Table S4 List of phage genomes used.
	(DOCX)
	Text S1 Probability to encounter traces of HR at random
	around mosaics.
	(DOCX)

Table S1 :

 S1 bacterial and phage strains used in this study.[invPL, 800bp IR, 78% id] : same as c, but inverted orientation of the sequences inside the IR, including PL. The genotype of this region is ea10:oxa7 rexA:(oxa5-cat-+) orf28-ral e. Refardt D, Rainey PB (2010) Tuning a genetic switch: experimental evolution and natural variation of prophage induction. Evolution 64: 1086-1097

	Strain	Genotype	Commentary
	MAC1117	MG1655 ilvD:cat	gift from S. Delmas (INSERM U1001, Paris,
			France)
	MAC1400	MG1655 stfR::cat	this study
	MD7	MG1655 stfR::cat recA306 srl::Tn10	this study
	MD19	MG1655 tfaQ::cat	this study
	MD63	MG1655 tfaQ::cat recA306 srl::Tn10	this study
	MD36	MG1655 ybcV::Kan	this study. Allele from Keio collection
	MD61	ybcV::Kan recA306 srl::Tn10	this study
	MD22	MG1655 yecD::cat	this study
	MD58	MG1655 nohD::cat	this study
	MD68	MG1655 ybcN::cat	this study
	MAC	C600 P2 lysogen	[19]
	MAC	C600 recA306 srl::Tn10	[19]
		Phage strains
	λMD 50	λPaPa	reference laboratory strain of phage λ . Gift
			from D. Refardt (Refardt & Rainey, 2010) e
	λMD51	λPaPa ble	this study
	Urλ	Urλ	Original isolates of λ. From the E. coli K12 strain
			of the E. Coli Genetic Stock Center.
	Urλ ble	Urλ ble	Urλ strain carrying the ble gene that confers
	SOC 27	Urλ ble redβ::FRT	resistance to phleomycine. gift from L. Le Chat this study
	SOC 28	Urλ ble exo::FRT	this study
	SOC 29	Urλ ble orf::FRT	this study
	SOC 30	Urλ ble rap::FRT	this study
	Φ80 MD2 Nec4	Φ80 ble [800bp IR, 100% id] a cI857ts b(22297-	this study [19]
	Nec1	27728) [invPL, 800bp IR, 100% id] b cI857ts	[19]
	Nec6	b(22297-27728) [800 bp IR, 78%id] c cI857ts b(22050-	[19]
	Nec3	26766) [invPL, 800 bp IR, 78%id] d cI857ts (22050-26766)	[19]
	Nec9 Nec10 Nec11 Nec12	[800bp IR, 100% id] cI857ts (22297-27728) red::recET [800 bp IR, 78%id] cI857ts (22050-26766) red:: recET [800bp IR, 100% id] cI857ts (22297-27728) red::erf-exo red:: erf-exo [800 bp IR, 78%id] cI857ts (22050-26766)	this study this study this study this study
	a. [800bp IR, 100% id] : recombination cassette composed of 800 bp-long inverted and 100% identical repeats
	flanking the pL promoter in its native orientation. The genotype of this region is ea10:oxa7 rexA:(oxa7-cat-+) orf28-ral

b. [invPL, 800bp IR, 100% id]: same as a., but inverted orientation of the sequences inside the IR, including PL.

c. [800bp IR, 78% id] : recombination cassette composed of 800 bp-long inverted and 78% identical repeats flanking the pL promoter in its native orientation. The genotype of this region is ea10:oxa7-5 rexA:(oxa5-7-cat-+) orf28-ral d.

Table S2 :

 S2 Differences between the published sequence of λ and the Urλ-ble phage used in this study.

	λPaPa	Ur-λble	orf	Change*
	NC_001416
	138	-G	IR cos-nu1	-1
	542	C -> T	nu1
	after 14267

Table S3 : primers used in this study . a Gene deletions or insertions stfR::cat

 S3

		CGCCGTCACATGCCGGGACCATTACCGTGTATGAAGATTCCAACTTTTGGCGAAAATGAG
		GTTGATGCCGTTCCTGCGCTGGAAGACGCTGACTGAGCCGACGGGAACTTCATTTAAATGGCG
	tfaQ::cat GTTATTTATTTCTGAACTCGGTCCGTTACCGGAAAATGTTACCTGTCCTCCTTAGTTCCTATTCC
		TATCCTTCACCCAGGCTGTGCCGTTCCACTTCTGAAACTCCCCTTCTGCCACTCATCGCAGTAC
	redβ::FRT GATCCCGGTACGCTGCAGGATAATGTCCGGTGTCATGCTGTCCTCCTTAGTTCCTATTCC
		CATTGCTCACCACCAGGTTGATATTGATTCAGAGGTATAAAACGAGTGTAGGCTGGAGCTGCTTC
	exo::FRT CTTGGATTCCTGAAACAGAAAGCCGCAGAGCAGAAGGTGGCAGCATGAACACAGGAACACTTAACGGC
		CCTACCCGGATATTATCGTGAGGATGCGTCATCGCCATTGCTCCCCTGTAGGCTGGAGCTGCTTCG
	orf::FRT	GGGAGAGGGAAGTCATGAAAAAACTAACCTTTGAAATTCGGCAGCATTACACGTCTTGAG
		CATGCAGCCCTGTCTCCCCATCTCGCTTTCCACTCCAGAGCCCATGGTCCATATGAATATCCTCC
	rap::FRT	GATGATGGCTAAACCAGCGCGAAGACGATGTAAAAACGATGGCAGCATTACACGTCTTGAG
		GTCATGCGGCCTCACTTCTGCTATTTCGCAGGTCTTTGAGCCATGGTCCATATGAATATCCTCC
	Φ80-ble	CAACAAAGAGATAAATCCAAGCCCGTTTTGTACGGGCTGTATGACCATGATTACGCCAAG
		ATGCAGGTAACGCAGCCAGAATTGTTGATTGGCTGATCGTTAAGTTGGGTAACGCCAGG
	yecD::cat CTTCCACGCTACGCACACGGGCGATGCGCGGGTAGATATGGTTGACAACTTTTGGCGAAAATGAG
		AAGACGCCTGTAGTGCCGCCAGCGCCGAGCAGCACAATAACAGTAGGAACTTCATTTAAATGGCG
	nohD::cat ATTGAGTACGAACGCCATCGACTTACGCGTGCGCAGGCCGATGCACAGGACAACTTTTGGCGAAAATGAG
		CACCTGCGATCCGCGACAGCACGAAAGTACAGAATGCGGTTTCCACCACTGGAACTTCATTTAAATGGCG
	ybcN::cat CTCCAGCTTGGGTAAAAGATGCTCTCAAACACACATATCTCGGTTATGAAGGAACTTCATTTAAATGGCG
		TCGGAGGTATGGCGTAACGACTGGATAGTGGTGATATCACCGGTTACGACACAACTTTTGGCGAAAATGAG
		Detection of excision of defective prophages
	Rac	CTCCAGCATGGTATAGCTGTCTTTAC
		CAGATTTCTTATGCTGGGCGTTCCG
	Qin	CGACAATACGCGCCACATAA AACGGCGAGTAAGTAGTACGC	described in [82]
	Dlp12	CAAAAGCCATTGACTCAGCAAGG
		ACGGATAAGACGGGCATAAATGA
		Activity of oriJ (rac), semi-qPCR
	gyrB	GTCGAAGTGGCGTTGCAGTG
	λ Q	AGCCTGCCAGGTGAGTACCG GAGTGCGGAAGATGCAAAGG	described in (Fogg et al, 2010) b
		TTAACAGTGCGTGACCAGG
	oriJ	GTTGAGTCAGGCGAAATCC
		CCACGGTAACCACAATCAC
		Identification of recombination junctions in λ hybrids
	Qin	GCTCATGCCCATTCTTTC
		ACGTTTCTGCGGCATAT
	Rac	ACTGGCGTACTGACGGATTC (rac 1)
		ACGTTTCTGCGGCATAT	(rac 2)
		ACGTTTCTGCGGCATATC	(rac 3)
	Dlp12	TTAGCATCCGCCCATTCAAC
		TGGTCAGTTCGAGCATAAGG
		Construction of recET and exo/erf λ
	recE	CGAGCTCCCGCTCGAGCTTTTGCGATTGGTGGTTGC
		GGAGGATGAAACTGGCGAAGTCGCA
	recT	CGAGCTCCATTACACCGCCAGGCTGAA
		CCGCTCGAGGGAGGCTGGGCTAAGGAATATGCAA
	exo/erf	GTTTGCCGCATGTGGCTTTG
		GGAGGCCATCAACAACGAAGCCC TG
	pKD4	TGATTGCGCCTACCCGGATATTATCGTGAGGATGCGTCATTCCTCCTTAGTTCCTATTCC
		CATTGCTCACCACCAGGTTGATATTGATTCAGAGGTATAATGCAGATTGCAGCAT TACAC
	a. Unless specified, primers were designed for this study.
	b. Fogg PC, Allison HE, Saunders JR, McCarthy AJ (2010). J Virol 84: 6876-6879

Table S4 : List of phage genomes used.

 S4

	Phi1 wV7	164270 166452		V V	T4 T4	NC_009821 NC_019505, *T4
	phi80 YYZ-2008	46150 54896		T T	 	JX871397 NC_011356
	Phage phiV10	length (bp) 39104	life style a T	family 15	accession number, *redundancy with NC_007804
	186 Qin	30624 20458		T D	P2 	NC_001317 NC_000913.2(1630450..1646830)
	536 86 rac a. D, defective; T, temperate; V, virulent. 46023 60238 23060 RB14 165429 FIGURE S1	T T D V	   T4	NC_008253(1189882..1228488) NC_008464 NC_000913.2(1409966..1433025) NC_012638, *T4
	933W RB15	61670 166877		T V	 T4	NC_000924 , *VT2Sakai phage.ggc.edu/gbrowse/RB15/RB15.fa, *T4
	BP-4795 RB16	57930 176788		T V	 T4	NC_004813 NC_014467
	Bp7 RB18	168066 166677		V V	T4 T4	NC_019500 phage.ggc.edu/gbrowse/RB18/RB18.fa, *T4
	CC31 RB26	165540 163036		V V	T4 T4	NC_014662 phage.ggc.edu/gbrowse/RB26/RB26.fa, *T4
	cdtI RB32	47021 165890		T V	 T4	NC_009514 NC_008515, *T4
	CP4-57 RB43	22030 180500		D V	P2 T4	NC_000913.2(2753978..2776007) NC_007023
	CP4-6 RB49	34306 164018		D V	fragments T4	NC_000913.2(262184..296489) NC_005066
	dlp12 RB51	21302 168394		D V	 T4	NC_000913.2(564025..585326) NC_012635, *T4
	e14 RB69	15193 167560		D V	 T4	NC_000913.2(1195443..1210646) NC_004928
	HK022 RTP	40751 46219		T V	 T1	NC_002166 NC_007603
	HK446 rv5	39026 137947		T V	 RV5	NC_019714 NC_011041
	HK620 sp1	38297 10586		T D	 unknown	NC_002730 NC_002695.1(300041..310626)
	HK97 sp10	39732 51112		T D	 	NC_002167 NC_002695.1(1921414..1972525)
	HX01 sp11	169158 45778		V D	T4 	NC_018855, *RB69 NC_002695.1(2158174..2203951)
	IAI39-Ph01 sp12	32853 46142		D D	 	NC_011750.1(532595..565447) NC_002695.1(2203952..2250093)
	IAI39-Ph02 sp13	12130 21120		D D	 P2	NC_011750.1(923080..935209) NC_002695.1(2592901..2614020)
	IAI39-Ph03 sp14	28321 44029		D D	 	NC_011750.1(1555796..1584116) NC_002695.1(2668007..2712035)
	IAI39-Ph04 sp15	18544 47879		D T	P2 	NC_011750.1(1882552..1901095) NC_002695.1(2895926..2943804)
	IAI39-Ph05 sp16	23629 8551		D D	 unknown	NC_011750.1(1932603..1956231) NC_002695.1(3192983..3201533)
	IAI39-Ph06 sp17	55581 24199		D D	unknown unknown	NC_011750.1(1992456..2048036) NC_002695.1(3475965..3500163)
	IAI39-Ph07 sp18	13835 38759		D D	 Mu	NC_011750.1(2060694..2074528) NC_002695.1(5040843..5079601)
	IAI39-Ph08 sp2	41562 12887		D D	15 P2	NC_011750.1(2751408..2792969) NC_002695.1(310627..323513)
	IAI39-Ph09 sp3	11389 38586		D D	unknown 	NC_011750.1(2934628..2946016) NC_002695.1(891123..929708)
	IAI39-Ph10 sp4	14803 49650		D D	Mu 	NC_011750.1(4625205..4640007) NC_002695.1(1161091..1210740)
	IAI39-Ph11 sp5	44948 62708		D T	 	NC_011750.1(5025091..5097038) NC_002695.1(1246012..1308719), *VT2Sakai
	IME08 sp6	172253 48423		V D	T4 	NC_014260 NC_002695.1(1541470..1589892)
	jk06 sp7	46072 15463		V D	T1 unknown	NC_007291 NC_002695.1(1594570..1610032)
	JS10 sp8	171451 46897		V D	T4 	NC_012741, *JS98 NC_002695.1(1618153..1665049)
	JS98 sp9	170523 58175		V D	T4 	NC_010105 NC_002695.1(1757506..1815680)
	JSE SPN3US	166418 240413		V V	T4 unknown	NC_012740, *RB49 JN641803
	K1-5 stx2-phage1717	44385 62147		V T	Sp6 	NC_008152 NC_011357
	K1E stx2-phageI	45251 61765		V T	Sp6 	NC_007637 NC_003525, *VT2Sakai
	K1F T1	39704 48836		V V	T7 T1	NC_007456 NC_005833
	KP15 T2	174436 163833		V V	T4 T4	NC_014036 http://phage.ggc.edu/gbrowse/T2/T2.fa, *T4
	KplE1 T3	9687 38208		D V	fragments T7	NC_000913.2(2464404..2474619) NC_003298
	lambda T4	48502 168903		T V	 T4	NC_001416 NC_000866
	Mu T4T	36717 168920		T V	Mu T4	NC_000929 http://phage.ggc.edu/gbrowse/T4T/T4T.fa
	N15 T5	46375 121750		T V	N15 T5	NC_001901 NC_005859, *T4
	N4 T6	70153 168705		V V	N4 T4	NC_008720 http://phage.ggc.edu/gbrowse/T6/T6.fa, *T4
	NJ01 T7	77448 39937		V V	C3 T7	NC_018835 NC_001604
	P1 TLS	94800 49902		T V	P1 T1	NC_005856 NC_009540
	P2 vB-EcoM-ACG-C40	33593 167396		T V	P2 T4	NC_001895 NC_019399, *T4
	P22 vB-EcoM-VR7	41724 169285		T V	P22 T4	NC_002371 NC_014792
	P27 VT2-Sakai	42575 60942		T T	unknown 	NC_003356 NC_000902
	P4 Wphi	11624 32684		T T	P2 P2	NC_001609 NC_005056

Homology region mean % identity N MEPS for Redβ (23 bp) N MEPS for RecA (31 bp)

	0.4
	0.3
	0.2
	0.1
	0
	15	30	45	60	75	90	105	120	135	150	165	180	195	210	225
										Dlp12-λ		98%		2211	2003
										Qin-λ		96%		271	218
										Rac-λ		88%		125	55
										yecD-Φ80		96%		156	118
										Dlp12-Φ80		79%		21	12
										oxa5/oxa7		78%		0	0

  showed that RecA E.coli can nucleate on SSB covered 48 nt ssDNA and drive away SSB E.coli by polymerization. Another possible inhibitor of RecA E.coli annealing in vivo is UvrD E.coli , a helicase known to disrupt the RecA E.coli

Table 2 :

 2 Oligonucleotides for in vivo recombineering and in vitro SSA assays. Red letters highlight the mismatches relative to the cat SS gene

	Name	N of	Divergenc	Sequence
		misma	e relative
		tches	to catss
	GO34	0	0 %	CAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATAGTATACGTAAGGCG
				ACAAGGTGCTGATGCCGCTGGCGATTCAGGT
	Map98	2	2 %	CAACTTCTTCGCCCCCGTTTTCACCATGGGCAAATATTATACGCAAGGCG
				ACAAGGTGCTGATGCCGCTGGCGATTCAGGT
	Map99	5	6 %	CAACTTCTTCGCGCCCGTTTTCACTATGGGCAAATATTATACGCAAGGCG
				ACAAGGTGCTGATGCCCCTGGCGATTCAGGT
	Map10	10	12 %	CAACTTTTTCGCGCCCGTCTTCACTATGGGCAAATATTATACGCAAGGCG
	0			ATAAGGTGCTCATGCCCCTGGCCATTCAGGT
	Map10	16	20 %	CAACTTTTTTGCGCCCGTCTTTACTATGGGGAAATATTATACGCAAGGGG
	1			ATAAGGTCCTCATGCCCCTCGCCATTCAGGT
	Map10	23	28 %	CAATTTTTTTGCGCCGGTCTTTACTATGGGGAAGTATTACACGCAAGGGG
	2			ATAAAGTCCTCATGCCCCTCGCCATCCAAGT

  . This property of annealing at the replication fork is taken into account when designing oligonucleotides for single Chapter IV: Discussion, Mosaic acquisition: causes and consequences, Phage recombinases, a major cause of mosaïcism 126 strand recombineering reactions in vivo. The aim of this technique is to anneal an oligonucleoide on the lagging strand template to bring a point mutation by assimilation of this oligonucleotide(Costantino, Court 2003). However, in the real life, Redβ and other recombinases of this super-family are able to recombine DNA fragments of much longer size than oligonucleotides. For example, in the Chapter II, we saw that several phage Rad52 recombinases are able to capture genes from the bacterial chromosome and incorporate them into the phage genome or recombine DNA from different phage genomes. How this happens exactly is not understood yet, but based on the idea that Redβ anneals at the replication fork, Poteete proposed a model of polymerase slippage where a ssDNA 3' overhang is wrapped around a Redβ ring and anneals on the lagging strand template of the replication fork. The polymerase of the leading strand slips to the annealed DNA and continues the DNA polymerization (see Figure6B in Chapter I,(Poteete 2008)). However, this model explains only half of the gene capture process as observed in Chapter II. Indeed, to complete the process, the replication fork has to perform two events of slippage promoted by Redβ in a range of 1 kb to capture a single gene.

  . Gp2.5 cannot invade a dsDNA and needs the phage replicative helicase Gp4 to open the dsDNA. Contrary to other phage recombinases, the capacity of Gp2.5 to recombine diverged sequences has not been tested. It will be interesting to test Chapter IV: Discussion, Mosaic acquisition: causes and consequences, Consequences on genomes 130 this capacity in our in vitro SSA assay involving diverged oligonucleotides as presented in Chapter III, and then in vivo with our ssDNA recombineering test as presented in Chapter III.

  To follow on the above reasoning, if phages use the Chapter IV: Discussion, Mosaic acquisition: causes and consequences, Consequences on genomes 131 faithful RecA, they should have backbone/VS structure. However, these prophages have a mosaic structure. There are at least two other ways for phages to exchange DNA:(1) to encounter a phage encoding a recombinase in the same bacterium, or (2) to capture genes from prophages in the host genome, as we showed in Chapter II that RecA promotes this type of events.It is to be noticed that genome comparisions show that virulent phages of the T4 family have a backbone/VS genome structure[START_REF] Comeau | Modular architecture of the T4 phage superfamily: a conserved core genome and a plastic periphery[END_REF][START_REF] Sullivan | Phylogenomics of T4 cyanophages: lateral gene transfer in the 'core' and origins of host genes[END_REF]). It may be, as discussed in Chapter II, that virulent phages encounter each other with a lower frequency than temperate phages, and so have less opportunities to exchange genetic material. In addition, the fidelity of UvsX recombinase is unknown at present.While the number and length of phage mosaics due to recent exchanges are now well explored, with an average of 2 mosaics per genome pair and a median mosaic length of 400 bp among temperate phage pairs (see Chapter II), we still do not have an overview on the particular genes that are being exchanged. This analysis can be performed with the data generated in the Chapter II. However,

phage genomes are poorly annotated, preventing clear conclusion. A global annotation work is currently in progress in the team (H. Delattre, O. Souai, M.A. Petit, personal communication).

B) A danger for host genomes

When temperate phages are integrated into the bacterial chromosome, almost all prophage genes are repressed by a phage encoded repressor. However, leaks of transcription lead to a weak expression of prophage recombinases (as other genes), which could affect the host genome stability.

  Could it be possible that the ancestral RecA was a core-only RecA? And RecA/UvsX and Rad51/RadA diverged from each other genetically, by the acquisition of different supplementary ssDNA binding domains, and have converged phenotypically, as suggested by[START_REF] Yang | Comparison of bacteriophage T4 UvsX and human Rad51 filaments suggests that RecA-like polymers may have evolved independently[END_REF])? A tree and an evolution history can be drawn with some duplication of genes/genomes to join all RecA-likes (Figure21and[START_REF] Lin | Origins and evolution of the recA/RAD51 gene family: evidence for ancient gene duplication and endosymbiotic gene transfer[END_REF])). A protein named KaiC, made of two RecA core domains in tandem, is homolog to aRadC, a core-only RadA in Archaea, which seems to branch very early in the RecA evolution tree(Haldenby, White, Allers 2009;[START_REF] Liang | Knockouts of RecA-like proteins RadC1 and RadC2 have distinct responses to DNA damage agents in Sulfolobus islandicus[END_REF]. The common ancestor to all Rad51like proteins seems to be a aRadC-like core-only RecA. The other core-only RecA, collectively designated as Radβ, are domesticated to serve Rad51/RadA (Radα), and Radβ is close to Radα[START_REF] Lin | Origins and evolution of the recA/RAD51 gene family: evidence for ancient gene duplication and endosymbiotic gene transfer[END_REF]. It would be interesting to investigate where Sak4 would branch in such tree. It may either be the last phage representative of this aRadC-like ancestor that has its own recombination process or branch closer to RecA like Radβ and Radα.

	Chapter IV: Discussion, Origin and distribution of recombinases, The phage Rad52 domesticated by
				Eukaryotes
	Archaea and Eukaryots, full size		Bacteria, full size
	Radα +Nt	aRadC/KaiC	RecA +Ct
	Radβ	core-only	Sak 4	Sak 4
		?
		?
		Ancestral
		RecA
		core-only ?
	2) The phage Rad52 domesticated by Eukaryotes
	Rad52-like recombinases are the predominant recombinases of phages. They were first classified into
	three groups: Redβ/RecT, Erf and Sak (Iyer, Koonin, Aravind 2002), and then clustered into one super
				135

1) The ubiquitous RecA RecA (Bacteria) and Rad51 (Eukaryotes) share a common domain, the core domain, capable of DNA binding and ATP hydrolysis, and have both a supplementary ssDNA-binding domain but not placed at the same position, in Ct for RecA and Nt for Rad51, which are not homologous to each other. However, UvsX (Phages) and RecA are perfectly homologous. This suggests that RecA and UvsX evolved from a common ancestor, and are more distantly related to Rad51. RadA (Archaea) is the perfect homolog of Rad51, with a similar Nt ssDNA binding domain

(Haldenby, White, Allers 2009)

.

family related to the Eukaryotic Rad52

(Lopes et al. 2010)

. No homologs were found in Archaea but Eukayotes have three Rad52 related proteins capable of promoting Rad51-independent SSA:

(1)

Rad52, having the Nt homologous to phage Rad52 and a supplementary Ct domain, that is a Rad51

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March 2014 | Volume 10 | Issue 3 | e1004181 65

Remerciements

Acknowledgments

We are grateful to Drs Plunkett and Kuzminov for communicating the sequence of phage W80 prior to its publication. We also thank Michael Dubow and Magali Leroy for the sequencing of the phages used in this study. We are also indebted to Ludovic Lechat for the gift of Urlble, and to David Halpern for the constructions of the orf and rap deletions. We thank A. Gruss, P. Serror and M. Ansaldi for critical reading of the manuscript prior to submission. Our special thanks go to Colin Tinsley for his constant support along this work, and his analysis of the IAI39 defective prophages.

ACKNOWLEDGEMENT

We thank Marianne De Paepe and Xavier Veaute for their insights all along this work and help with the manuscript, and Hadrien Delattre for his help with gene context analysis.

FUNDING

This work was supported by the Agence Nationale de la Recherche [Dynamophage contract].

This work was performed with the financial support of Fondation pour la Recherche Me ´dicale (MDP) and Agence Nationale de

TABLE AND FIGURES LEGENDS Table 1 : Strains strain genetic background plasmid recombinational genes on plasmid Origin Map1798 Map1798 pJA17 hkaM HK620 sak4 HK620 ssb HK620 abc2 HK620 This study Map1801 Map1798 pKD46 orf60a λ redα λ redβ λ gam λ (34) Map1802 AB1157 recA mutL cat ss none This study Map1879 Map1798 pJA4 none (Lopes et al., 2010) Map1881 Map1798 pGH3 sak4 HK620 This study Map1883 Map1798 pJA12 sak4 PA73 (Lopes et al., 2010) Map1894 Map1798 pJA12 redβ λ This study Map1895 Map1798 pGH19 sak4 HK620 ssb HK620 This study Map1897 Map1798 pGH20 sak4 HK620 0 abc2 HK620 This study Map1922 Map1798 pGH21 ssb HK620 This study Map1931 Map1798 pGH24 sak4 PA73 ssb PA73 This study

Abbreviation list µM microMolar

Chapter II:

Temperate phages acquire DNA from defective prophages by relaxed homologous recombination: the role of rad52-like recombinases.

Abstract

Bacteriophages (or phages) dominate the biosphere both numerically and in terms of genetic diversity. In particular, genomic comparisons suggest a remarkable level of horizontal gene transfer among temperate phages, favoring a high evolution rate. Molecular mechanisms of this pervasive mosaicism are mostly unknown. One hypothesis is that phage encoded recombinases are key players in these horizontal transfers, thanks to their high efficiency and low fidelity. Here, we associate two complementary in vivo assays and a bioinformatics analysis to address the role of phage encoded recombinases in genomic mosaicism. The first assay allowed determining the genetic determinants of mosaic formation between lambdoid phages and Escherichia coli prophage remnants. In the second assay, recombination was monitored between sequences on phage l, and allowed to compare the performance of three different Rad52-like recombinases on the same substrate. We also addressed the importance of homologous recombination in phage evolution by a genomic comparison of 84 E. coli virulent and temperate phages or prophages. We demonstrate that mosaics are mainly generated by homology-driven mechanisms that tolerate high substrate divergence. We show that phage encoded Rad52-like recombinases act independently of RecA, and that they are relatively more efficient when the exchanged fragments are divergent. We also show that accessory phage genes orf and rap contribute to mosaicism. A bioinformatics analysis strengthens our experimental results by showing that homologous recombination left traces in temperate phage genomes at the borders of recently exchanged fragments. We found no evidence of exchanges between virulent and temperate phages of E. coli. Altogether, our results demonstrate that Rad52-like recombinases promote gene shuffling among temperate phages, accelerating their evolution. This mechanism may prove to be more general, as other mobile genetic elements such as ICE encode Rad52-like functions, and play an important role in bacterial evolution itself. Poisson distribution and found to be in the 10 -14 -10 -16 range for all groups of phages tested (values reported in Table 1 of the main text). We conclude therefore that the number of traces of HR found next to the mosaics is highly significant. As a control, the same study was done on the hits corresponding to IS. 71 among 533 hits analysed contained a trace of homologous recombination at their boundary, for a random number expected Nr of 46. In this case, a p-value of 0.025, at the limit of significativity, was obtained, as expected for IS that should not exchange by HR but by transposition (unless degenerated).

Author Contributions

Chapter III:

Sak4, a phage encoded core-only RecA, needs its cognate SSB to perform single strand annealing in vivo.

ABSTRACT

Bacteriophages, the viruses infecting bacteria, are remarkable under many aspects, one of them being the incredible diversity of proteins they encode to perform DNA replication and homologous recombination processes. Looking back at these extremely simplified forms of life may help to untangle how similar proteins work in more sophisticated organisms. In a previous study apprehending phage recombinase diversity, we uncovered the Sak4 family, which are core-only RecA-like proteins. Such proteins are not found in Bacteria, but are present in Archaea (RadB), and in Eukaryota (CSM2 and PSY3 of the Shu-complex in yeast, XRCC2 in human), where they participate in recombination events by helping the nucleation/polymerization of Rad51/RadA filaments. But none of these core-only RecA seems to directly promote some sort of recombination by itself. Here, we report a biochemical and genetic characterization of two Sak4, encoded by phages HK620 of Escherichia coli and PA73 of Pseudomonas aeruginosa. We show that a distant homolog of SSB is present next to the sak4 gene in most phages. Using a ssDNA recombineering assay, we found that Sak4 HK620 promotes SSA in vivo, provided its cognate SSB is co-expressed with it. Similarly to Redβ λ , Sak4 HK620 is active on diverged DNA substrates, up to 12% in vivo. In vitro, Sak4 proteins were insoluble, except for the GST-Sak4 PA73 fusion, which was further studied. It was unable to perform the typical, RecA-dependent strand exchange reaction, but performed efficient SSA, pairing up to 20% diverged oligonucleotides. Its cognate SSB had affinity to ssDNA, but did not enhance any of its biochemical properties.

strains deleted for recA. Some evidence has nevertheless been gathered, suggesting that the Sak protein of the Lactococcus lactis phage ul36 also facilitates RecA-dependent strand exchange in vitro (Ploquin et al., 2008), and that the Redβ protein of phage λ is accessory to RecA-dependent recombination in vivo, under conditions where the phage is not replicating (Stahl et al., 1997).

Besides this important and particularly well studied family of phage recombinases, comparative genomics on phage genomes permitted us to detect two other important superfamilies of recombinases involved in homologous recombination (Lopes et al., 2010). One is composed of proteins similar to Gp2.5 of phage T7, infecting E. coli. This recombinase has an OB-fold, as the single strand binding (SSB) proteins, with a supplementary α-helix (Hollis et al., 2001;Rezende et al., 2003):

it functions both as an SSB and a recombinase in vitro and in vivo, and specific mutations can alter independently each of these two activities (Rezende et al., 2003). The other superfamily includes recombinases similar to RecA, and is subdivided into two groups: (i) the full-size RecA-like proteins, such as UvsX of phage T4 infecting E. coli [START_REF] Liu | Assembly and dynamics of the bacteriophage T4 homologous recombination machinery[END_REF]for review), that are mostly found in some virulent phages with large genomes. These recombinases function in a way similar to RecA or Rad51 (Liu et al., 2011). recombination gene, it was supposed that Sak4 was a recombinase (Bouchard and Moineau, 2004).

Indeed, using a single strand recombineering assay, we reported that the Sak4 protein encoded by phage PA73 of P. aeruginosa promoted a low level of SSA (Lopes et al., 2010).

Some other core-only RecA are present in Archaea, where they are designated as RadB, and in Eukaryota, where the most studied are the Csm2 and Psy3 proteins of the Shu-complex in Saccharomyces cerevisiae, and the Xrcc2 protein in human. These proteins participate in recombination events by stabilizing or helping the nucleation/polymerization of Rad51 or RadA filaments (Komori et al., 2000;[START_REF] Godin | The Shu complex interacts with Rad51 through the Rad51 paralogues Rad55-Rad57 to mediate error-free recombination[END_REF]. But none of these core-only RecA seems to be a recombinase by itself, able to directly promote strand invasion or SSA. However, the BCDX2 complex,

Supplementary material

Choice of the recombineering assay

The recombineering assay that we conducted previously (1) was based on the selection of Rifampicin resistant (RifR) colonies produced by the recombineering of a 51nt-long oligonucleotide complementary to the rpoB gene. Several improvements were made in the present study. In the former protocol, competent cells were prepared in large batches, frozen at -80°C in aliquots, and used repeatedly. In addition, once electroporated and let grown 1hour for expression, cells were further incubated overnight on the bench, prior to titrations on plates. These two factors proved on the long range to be responsible for variations in cell viability, and hence estimations of recombineering efficiencies were not always reproducible. Following the protocol recommended by Dr Court, competent cells were prepared freshly, and the overnight incubation on the bench was suppressed. However, this last change reduced drastically the yield of RifR colonies, suggesting that 1h expression time was not sufficient to allow the plain recovery of recombinants. To increase the expression time without letting grow cells in liquid (as expected if the expression time is extended), cells were plated after the 1h expression period embedded into top agar on non-selective LB plates, and let grown 2 more hours at 37°C. At this stage only, the rifampicin selection was applied, by a topagar overlay. This (old) technique proved to increase by a factor of 100 the yield of RifR colonies. The top-agar technique also permitted a better recovery of viable cells (that were in the range of 10 9 -/ml, rather than 10 8 otherwise). However, this new protocol led to an unexpectedly high "background" of recombineering with the recombinase-less, control plasmid pJA4: this level, which was estimated at 5 10 -7 spontaneous recombinants per viable cell in Lopes et al., reached now 2 10 -6 . This high background hindered the detection the recombineering activity of the Sak4 PA73 recombinase (1. 5 10 -6 in Lopes et al., 2 10 -6 here).

To reduce this background level, and also in perspective of the experiments we wanted to conduct in a mutS background (to use degenerated oligonucleotides), a new selection system was constructed.

Based on the cat gene, two points were mutated to introducing stop codons, next to each other in cat gene (strain MAC1802, see construction details below). The cat cassette (together with a KanR marker gene) was introduced at the vicinity of rpoB, within the thi operon, so as to delete thiE and thiF (the thiE gene is already mutated in AB1157). Spontaneous recombineering at cat was low (5 10 -

9

). However, recombineering with pKD46 was reduced by a factor of 200 at this locus, compared to the rpoB locus: 4.8 10 -6 CmR/viable cell, for 10 -3 RifR/viable cell. Comparison of recombineering efficiencies obtained at these two loci is shown in the Table below. Another set of recombineering assays were performed into strain G205, a derivative of HME57 (a strain used in the Court laboratory, which is galK) rendered recA, so as to compare recombineering efficiencies at the rpoB and galK loci, in a MutS+ background (both oligonucleotides generate single C:C mispairs, that are not recognized by MutS, ( 3)). For the Gal + selection, no top agar overlay is used (to follow Court's lab protocol), and viable cells are counted on the same M9 agar medium, supplemented with glucose 0.2%.

Supplementary references

Supplementary tables

Supplementary Table 1: Oligonucleotides for plasmid and strain constructions. name sequence

Supplementary Figure 1: Genome context for the SSB HK620 protein: Phage homologs of SSB HK620 (in red) were searched using HHsearch, and those retrieved with a 95% probability cutoff are shown. The same search was performed for the 4 genes left and right off all SSB homologs that had been retrieved, to inspect local co-occurences. Sak4 homologs are coloured in yellow. Under the figure are the annotation taken from PFAM, ACLAME or GENBANK.

Suplementary Figures

Supplementary Figure 2: Sequence analysis of ~10 recombinants obtained by ssDNA recombineering in vivo, with Redβ (upper, blue sequences) and Sak4 HK620 (lower, yellow sequences) using oligonucleotide map100 (12% diverged, relative to the catss sequence). On top of the figure is the sequence of map100 with the central T and C in bold letters showing the two mismatches permitting correction of the 2 stop codons in catss. The red bases correspond to silent mutations introduced into map100 to create a 12% diverged sequence, relative to catss. + indicates mutations present in map100 that have been incorporated into the CmR recombinants obtained, -: mutations that were not incorporated.