Al C Citti
email: c.citti@envt.fr

Citti

E Dordet-Frisoni

L X Nouvel

C H Kuo

E Baranowski

Horizontal Gene Transfers in Mycoplasmas

The class Mollicutes (trivial name "mycoplasma") is composed of wall-less bacteria with reduced genomes whose evolution was long thought to be only driven by gene losses. Recent evidences of massive horizontal gene transfer (HGT) within and across species provided a new frame to understand the successful adaptation of these minimal bacteria to a broad range of hosts. Mobile genetic elements are being identified in a growing number of mycoplasma species, but integrative and conjugative elements (ICEs) are emerging as pivotal in HGT. While sharing common traits with other bacterial ICEs, such as their chromosomal integration and the use of a type IV secretion system to mediate horizontal dissemination, mycoplasma ICEs (MICEs) revealed unique features: their chromosomal integration is totally random and driven by a DDE recombinase related to the Mutator-like superfamily. Mycoplasma conjugation is not restricted to ICE transmission, but also involves the transfer of large chromosomal fragments that generates progenies with mosaic genomes, nearly every position of chromosome being mobile. Mycoplasmas have thus developed efficient ways to gain access to a considerable reservoir of genetic resources distributed among a vast number of species expanding the concept of minimal cell to the broader context of flowing information.

Introduction

Horizontal gene transfer (HGT) is a major contributor to microbial evolution and adaptation by allowing bacteria to rapidly acquire new traits from external resources. Mechanisms underlying this phenomenon include (i) natural transformation, with the uptake of naked DNA, (ii) transduction with the injection of viral DNA and (iii) conjugation, with conjugative elements as vessels navigating between cells [START_REF] Frost | Mobile genetic elements: the agents of open source evolution[END_REF][START_REF] Ochman | Lateral gene transfer and the nature of bacterial i n n o v a t i o n[END_REF]. Network analyses of gene sharing among bacterial genomes suggest that most HGT occurs when donor and recipient are proximate, and hence designate conjugation as the predominant mechanism of HGT [START_REF] Halary | Network a n a l y s e s s t r u c t u r e g e n e t i c d i v e r s i t y i n independent genetic worlds[END_REF][START_REF] Kloesges | N e t w o r k s o f g e n e s h a r i n g a m o n g 3 2 9 proteobacterial genomes reveal differences in lateral gene transfer frequency at different phylogenetic depths[END_REF].

Bacterial conjugation is a contact-dependent process actively transferring DNA from a donor, usually carrying a conjugative element, to a recipient cell most often lacking that element. Conjugative systems are encoded by plasmids but chromosomal Integrative and Conjugative Elements (ICE) have been recently recognized as the most abundant conjugative elements in prokaryote [START_REF] Guglielmini | The repertoire of ICE in prokaryotes underscores the unity, diversity, and ubiquity of conjugation[END_REF]. ICEs are self-transmissible Mobile Genetic Elements (MGE) that encode the machinery for their excision from the host chromosome, their transfer through a conjugative channel and subsequent integration into the recipient where they replicate as a part of the host chromosome [START_REF] Bellanger | Conjugative and mobilizable genomic islands in bacteria: evolution and diversity[END_REF]. Conjugative plasmids and ICEs most likely have an intricate, common evolution history but differ by their intracellular versus chromosomal residence. Bacterial genomes are also populated with integrative mobilizable elements (IME) that are not self-transmissible per se but can take advantage of other, resident conjugative machineries [START_REF] Daccord | Integrating conjugative elements of the SXT/ R391 family trigger the excision and drive the mobilization of a new class of Vibrio genomic islands[END_REF]. cells but can also involve chromosomal DNA regions as initially observed by [START_REF] Lederberg | Gene recombination in Escherichia coli[END_REF][START_REF] Lederberg | Gene recombination in Escherichia coli[END_REF][START_REF] Lederberg | Gene recombination in Escherichia coli[END_REF]. Using high-frequency recombination (Hfr) E. coli strains, these authors showed the horizontal transfers of genomic DNA, now known as Hfr-or oriT-based transfers. These events are initiated from an integrated origin of transfer (oriT) and are characterized by a gradient, with genes closer to the oriT being more reliably and more frequently transferred [START_REF] Hochhut | Mobilization of plasmids and chromosomal DNA mediated by the SXT element, a constin found in Vibrio cholerae O139[END_REF], mainly because of physical constraints applying on large molecules during transfer. More recently, chromosomal transfers have been described in Mycobacterium smegmatis [START_REF] Gray | Distributive conjugal transfer in mycobacteria generates progeny with meiotic-like genome-wide ! 18 mosaicism, allowing mapping of a mating identity locus[END_REF][START_REF] Derbyshire | Distributive conjugal transfer: New insights into horizontal gene transfer and genetic exchange in Mycobacteria[END_REF] and in Mycoplasma agalactiae [START_REF] Dordet-Frisoni | Chromosomal transfers in mycoplasmas: when minimal genomes go mobile[END_REF]) that differ from the classical oriTbased conjugation and thus are not directly associated to MGEs (see also below). In both cases, resulting transconjugants displayed chimeric genomes (see below). In M. smegmatis the situation was extreme, with some transconjugants having acquired up to 25% of the donor chromosome distributed over 30 loci and the associated mechanism was called Distributive Conjugal Transfer (DCT).

In this review, we described a new family of selftransmissible ICEs discovered in mycoplasmas (MICEs) and the occurrence in these minimal b a c t e r i a o f c h r o m o s o m a l t r a n s f e r v i a a n unconventional mechanism that resembles DCT. We further discussed the impacts of these transfers on mycoplasma evolution and on the emergence of new phenotypes with modified virulence traits when pathogenic species are concerned.

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Mollicutes (mycoplasmas) and the theory of reductive evolution

Mycoplasmas as minimal cells

Organisms of the Class Mollicutes belong to a large group of wall-less bacteria which taxonomy comprises six families and over 10 genera (see Table 1). In this review, we have mainly concentrated on three genera, which are Mycoplasma, Ureaplasma and Spiroplasma, and have used the trivial term "mycoplasma" to designate any member of the Mollicutes.

From a phylogenetic point of view, mycoplasmas have derived from Gram positive bacteria with low GC content by a process called reductive evolution, which was marked by successive and drastic genetic losses. As a result, current mycoplasmas are characterized by a small genome (ca. 0.5 to 2.0 Mbp), the total lack of a cell-wall and reduced metabolic pathways. With the exception of phytoplasmas (Table 1) and a few Mycoplasma species, most mycoplasmas are able to selfreplicate under laboratory conditions in axenic, complex media. Of note, bacteria with smaller genomes (genome size of ca. 0.1 to 0.7 Mbp) have b e e n i d e n t i f i e d b u t t h e s e a r e o b l i g a t e endosymbionts that do not seem to be able to survive outside of their host cells [START_REF] Mccutcheon | Extreme genome reduction in symbiotic bacteria[END_REF][START_REF] Moran | The tiniest tiny genomes[END_REF]. Because of their small size, mycoplasma genomes were among the first to be fully sequenced, starting in 1995 with the human pathogen Mycoplasma genitalium that displays the smallest gene-set known to support autonomous life. For the very same reason, these minimal bacteria have been used as a blue print for generating the first bacterial cell controlled by a chemically synthesized genome, M. mycoides JCVI-syn1.0 [START_REF] Gibson | Creation of a bacterial cell controlled by a chemically synthesized genome[END_REF].

Mycoplasmas as minimal pathogens

Despite a limited genetic content, a large number of mycoplasmas have emerged and are emerging as important pathogens of man, animals, insects and plants. In human and veterinary medicine, several Mycoplasma and Ureaplasma species are recognized pathogens, often associated with slowly progressive or chronic diseases [START_REF] Razin | Highlights of mycoplasma research--an historical perspective[END_REF][START_REF] Citti | Mycoplasmas and their host: emerging and re-emerging minimal pathogens[END_REF]. Most of these have a predilection for the mucosal of the respiratory and the genital tract, causing respiratory diseases and genital disorders, or for the joint, causing arthritis. These pathogenic species have developed common strategies to colonize and circumvent their immunocompetent hosts yet there is no correlation between their phylogenetic position (Figure 1) and a particular degree of virulence or host. For instance, the Hominis group includes species that are pathogenic either for ruminants such as M. agalactiae responsible of mastitis, pneumonia and arthritis in sheep and goat, or for swine such as M. hyopneumoniae responsible of enzootic pneumonia in pig, or for poultry with M. synoviae, that is associated to synovitis and/or airsacculitis. A similar observation can be made for the Pneumoniae or for the Spiroplasma groups. This latter group is remarkable because it includes several major ruminant pathogens, such as M. mycoides subsp. mycoides or M. capricolum capripneumoniae, along with Spiroplasma species [START_REF] Gasparich | The genus Spiroplasma and its non-helical descendants: phylogenetic classification, correlation with phenotype and roots of the Mycoplasma mycoides clade[END_REF]) that are pathogenic for plants and/or insects. For instance, S. citri and S. kunkelli of the Citri clade are two insect-transmitted phytopathogens, responsible of the citrus stubborn and the corn stunt diseases, respectively. The Apis clade includes several species pathogenic for insects such as S. apis for honeybees or S. culicicola for mosquitoes. Several species across multiple clades are pathogenic for aquatic crustaceans such as S. penaei [START_REF] Nunan | Spiroplasma penaei sp. nov., associated with mortalities in Penaeus vannamei, Pacific white shrimp[END_REF] of the Citri clade for saltwater shrimps and S. eriocheiris [START_REF] Wang | Spiroplasma eriocheiris sp. nov., associated with mortality in the Chinese mitten crab, Eriocheir sinensis[END_REF] of the Mirum clade for freshwater crabs. For more intimate associations with their hosts, some Spiroplasma lineages have become vertically-transmitted reproductive parasites with male-killing effects. This is the case of the Drosophila-associated S. poulsonii (Citri clade) and at least one unnamed lineage in the Ixodetis clade infecting ladybird beetles [START_REF] Tinsley | A new male-killing parasitism: Spiroplasma bacteria infect the ladybird beetle Anisosticta novemdecimpunctata (Coleoptera: Coccinellidae)[END_REF]. Interestingly, some Spiroplasma spp. have evolved as defensive mutualists providing their Drosophila hosts with a protection against nematodes [START_REF] Jaenike | Adaptation via symbiosis: recent spread of a Drosophila defensive symbiont[END_REF] or parasitic wasps (Xie et al. 2010(Xie et al. , 2011)). Finally, only one Spiroplasma species is known as a pathogen of vertebrates [START_REF] Tully | Interaction of spiroplasmas with plant, arthropod, and animal hosts[END_REF] namely S. mirum, the suckling mouse cataract agent [START_REF] Tully | Revised taxonomy of the class Mollicutes: proposed elevation of a monophyletic cluster of arthropodassociated mollicutes to ordinal rank (Entomoplasmatalesord. nov.), with Provision for familial rank to separate species with nonhelical morphology (Entomoplasmataceaefam. nov.) from helical species (Spiroplasmataceae), and emended descriptions of the order Mycoplasmatales, family Mycoplasmataceae[END_REF]).

Because of their limited coding capacity, most mycoplasmas have adopted a parasitic life-style and live in intimate contact with their hosts in which they often act as facultative intracellular symbionts.

Recently, Mollicutes-related endobacteria (MRE) have been described as representing a sister clade of the Mycoplasmatales and Entomoplasmatales, within the Mollicutes [START_REF] Naumann | The obligate endobacteria of arbuscular mycorrhizal fungi are ancient heritable components related to the Mollicutes[END_REF][START_REF] Naito | Minimal genomes of mycoplasma-related endobacteria are plastic and contain host-derived genes for sustained life within Glomeromycota[END_REF][START_REF] Torres-Cortés | Mosaic genome of endobacteria in arbuscular mycorrhizal fungi: Transkingdom gene transfer in an ancient mycoplasma-fungus association[END_REF]. These organisms are obligate symbionts of arbuscular mycorrhizal fungi (AMF), which have themselves formed intimate, mutualistic symbioses with the vast majority of land plants, providing them with mineral ! 5

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nutrients and water, receiving in return carbohydrates from photosynthesis. These MRE are found in most AMF species, indicating that their associations have an ancient origin. However, the biological functions of MRE are unknown.

Horizontal gene transfers in mycoplasmas

For decades, genome streamlining was thought to be the only force driving mycoplasma evolution. Indeed, HGT was thought to be marginal in these organisms because of their limited content in efficient recombination systems (Rocha et al. 2005) and in prophage or conjugative elements as means for DNA uptake (Sirand-Pugnet et al. 2007a, b). Interest in mycoplasmas HGT was revived by the advances in sequencing: comparative genomics revealed that large chromosomal DNA exchanges have taken place between phylogenetically remote mycoplasma species known to colonize the same habitat (Sirand-Pugnet et al. 2007b;[START_REF] Pereyre | Life on arginine for Mycoplasma hominis: clues from its minimal genome and comparison with other human urogenital mycoplasmas[END_REF]. The concomitant detection of putative ICEs [START_REF] Calcutt | Molecular genetic analysis of ICEF, an integrative conjugal element that is present as a repetitive sequence in the chromosome of Mycoplasma fermentans PG18[END_REF][START_REF] Marenda | A new integrative conjugative element occurs in Mycoplasma agalactiae as chromosomal and free circular forms[END_REF]) in these species further raised the prospect that these simple bacteria might be able to conjugate. Addressing this issue in mycoplasmas that are fastidious to grow and genetically difficult to manipulate was not trivial and only two early works were supportive of this hypothesis. The first one was based on phenotypic observations of resistance transfer in S. citri mutants [START_REF] Barroso | Chromosomal gene transfer in Spiroplasma citri[END_REF] and the second described a DNase-resistant transfer of transposon in M. pulmonis [START_REF] Teachman | Gene transfer in Mycoplasma pulmonis[END_REF]. The confirmation that modern mycoplasmas have retained a form of sexual competence and are able to exchange DNA via conjugation further came from the study of ICEs in ruminant mycoplasma species, using the ruminant pathogen M. agalactiae as model .

Mycoplama Integrative Conjugative Elements (MICEs): a new family of self-transmissible element with conjugative properties

Mycoplasma ICEs (MICEs) belong to the large family of bacterial ICEs also designated as conjugative transposons. Over the last decades, ICEs have been increasingly appreciated for their role in HGT [START_REF] Burrus | Shaping bacterial genomes with integrative and conjugative elements[END_REF][START_REF] Delavat | The hidden life of integrative and conjugative elements[END_REF] and their importance in the evolution of microorganisms is underlined by their wide distribution across diverse bacteria [START_REF] Wozniak | Integrative and conjugative elements: mosaic mobile genetic elements enabling dynamic lateral gene flow[END_REF][START_REF] Guglielmini | The repertoire of ICE in prokaryotes underscores the unity, diversity, and ubiquity of conjugation[END_REF]). These genetic elements are defined by two features: their integration in the host-chromosome and their capacity to encode a type IV secretion system that mediates their transfer from donor to recipient cells via conjugation [START_REF] Johnson | Integrative and Conjugative Elements (ICEs): What they do and how they work[END_REF]. One of the best-known ICEs is Tn916, which is a promiscuous transposon of Enterococcus fecalis that confers resistance to antimicrobials [START_REF] Roberts | Tn916-like genetic elements: a diverse group of modular mobile elements conferring antibiotic resistance[END_REF] and has been introduced by transformation in a number of mycoplasmas [START_REF] Voelker | Gene transfer in Mycoplasma arthritidis: transformation, conjugal transfer of Tn916, and evidence for a restriction s y s t e m r e c o g n i z i n g A G C T[END_REF][START_REF] Clewell | Conjugative transposon Tn916 and related elements[END_REF]. Whether Tn916 can self-disseminate horizontally in natural conditions to and among mycoplasmas is not known but several genital mycoplasmas resistant to tetracycline possess a tet(M) determinant displaying high identities (~97 %) to that carried by the Streptococcus pneumoniae Tn916 [START_REF] Mardassi | Evidence for the predominance of a single tet(M) gene sequence type in tetracycline-resistant Ureaplasma parvum and Mycoplasma hominis isolates from Tunisian patients[END_REF][START_REF] Calcutt | An excisioncompetent and exogenous mosaic transposon harbors the tetM gene in multiple Mycoplasma hominis lineages[END_REF].

MICEs gene content and organisation

MICEs were first described in two mycoplasma species belonging to the Hominis phylogenetic group (Figure 1): M. fermentans, an opportunistic human pathogen and M. agalactiae, an important pathogen of small ruminants. M. fermentans ICE, ! 7

Figure 1. Evolutionary relationships of selected mycoplasmas (Mollicutes) and MICE distribution The phylogenetic tree was inferred from 16S rDNA sequences using the Neighbour-Joining method [START_REF] Saitou | The neighbor-joining method: a new method for reconstructing phylogenetic trees[END_REF]. Phylogenetic clades commonly used in this manuscript are indicated on the left and the four main phylogenetic groups are indicated on the right side. The bootstrap consensus tree was inferred from 500 replicates and is taken to represent the evolutionary history of the taxa analysed [START_REF] Felsenstein | Estimating effective population size from samples of sequences: a bootstrap Monte Carlo integration method[END_REF]. Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (500 replicates) are shown next to the branches [START_REF] Felsenstein | Estimating effective population size from samples of sequences: a bootstrap Monte Carlo integration method[END_REF]. The evolutionary distances were computed using the Maximum Composite Likelihood method [START_REF] Tamura | Prospects for inferring very large phylogenies by using the neighbor-joining method[END_REF]) and are in the units of the number of base substitutions per site. The analysis involved 55 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 1159 positions in the final dataset. Evolutionary analyses were conducted in MEGA7 [START_REF] Kumar | MEGA7: Molecular evolutionary genetics analysis version 7 . 0 f o r b i g g e r d atasets[END_REF]. The distribution of MICE or MICE factors is indicated on the right side: full orange dots indicate species for which ICE were previously described in at least one strain; hatched orange dots designate species for which both CDS5 and CDS17 were concomitantly detected; white dots correspond to species for which no MICE has been reported or for which the concomitant occurrence of CDS5 and CDS17 was not detected. CDS5 and CDS17 were searched by blast analysis using standard parameters and CDS5 and CDS17 amino acid sequences of M. agalactiae (MAGA7030 and MAGA6970 respectively) and M. mycoides subsp. capri GM12 (MMCAP2_0555 and MMCAP2_0567, respectively), all available at the NCBI public database (https://www.ncbi.nlm.nih.gov/).

ICEF, [START_REF] Calcutt | Molecular genetic analysis of ICEF, an integrative conjugal element that is present as a repetitive sequence in the chromosome of Mycoplasma fermentans PG18[END_REF] and M. agalactiae ICE, ICEA, [START_REF] Marenda | A new integrative conjugative element occurs in Mycoplasma agalactiae as chromosomal and free circular forms[END_REF]) are very similar, large modular elements of approximately 20-30 kbp (Figure 2), occurring as multiple copies in their hostgenomes. Both MICEs are composed of about 20 structural genes with only three (namely cds5, cds12 and cds17) having predicted functional domains common to proteins with DNA mobilityrelated functions in other bacteria (Alvarez-Martinez and Christie 2009). These are (i) a TraG/VirD4 homolog (CDS5, pfam accession number PF02534) that usually couples the relaxome to the conjugal mating pore [START_REF] Balzer | Essential motifs of relaxase (TraI) and TraG proteins involved in conjugative transfer of plasmid RP4[END_REF][START_REF] Llosa | Bacterial conjugation: a two-step mechanism for DNA transport[END_REF], (ii) a single stranded DNA binding (SSB) protein (CDS12, pfam accession number PF00436) and (iii) a TraE/VirB4 homolog (pfam accession number PF05309) usually involved in DNA transport [START_REF] Komano | The transfer region of IncI1 plasmid R64: similarities between R64 tra and Legionella icm/ dot genes[END_REF][START_REF] Rabel | The VirB4 family of proposed traffic nucleoside triphosphatases: Common motifs in plasmid RP4 TrbE are essential for conjugation and phage adsorption[END_REF].

With the advances in genome sequencing, several other MICEs have since been described which overall organization is relatively similar [START_REF] Calcutt | Molecular genetic analysis of ICEF, an integrative conjugal element that is present as a repetitive sequence in the chromosome of Mycoplasma fermentans PG18[END_REF][START_REF] Pinto | Molecular analysis of an integrative conjugative element, ICEH, present in the chromosome of different strains of Mycoplasma hyopneumoniae[END_REF][START_REF] Nouvel | Comparative genomic and proteomic analyses of two Mycoplasma agalactiae strains: clues to the macro-and micro-events that are shaping mycoplasma diversity[END_REF][START_REF] Wise | Complete genome sequence of Mycoplasma bovis type strain PG45 (ATCC 25523)[END_REF][START_REF] Thiaucourt | Mycoplasma mycoides, from "mycoides Small C o l o n y " t o " capri" . A m i c r o e v o l u t i o n a r y perspective[END_REF][START_REF] Shu | Complexity of the Mycoplasma fermentans M64 genome and metabolic essentiality and diversity among mycoplasmas[END_REF] as illustrated in Figure 2 In other bacteria, the global ICE structure consists of three modules that enable its maintenance, dissemination, and regulation. Little is known regarding the functionality of MICE components but the clustering of seven genes encoding putative proteins with transmembrane domains (in ICEA CDS5 to CDS19) most likely represents the module associated with the conjugative channel (see also below Figure 3).

Blast searches also revealed the presence of shorter and putatively degraded MICEs in mycoplasma genomes, with some being limited to two intact genes (see Figure 2, ICEA5632-IV). As shown in Figure 2, they often carry pseudogenes that most likely reflect past MICE attacks followed by MICE erosion and are thus commonly refer as MICE vestige (vMICE). For instance, diverse vMICEs have been detected in M. agalactiae with strain 5632 having two short vMICEs (<5 kbp) in addition to the three complete ICEA copies and PG2 having a longer vestige (ca. 22 kbp), vICE-PG2, composed of numerous pseudogenes. Several vMICEs, such as ICEB-2 of M. bovis, also harbor IS elements, with some being inserted in MICE backbone genes. The accumulation of disruptive mutations and IS supports the assumption of vMICE being non-functional and slowly decaying. Indeed, experimental studies have shown that vICE-PG2 is not active and is not horizontally transferred under laboratory conditions (Dordet-Frisoni et al. 2013b). Nevertheless, the occurrence of vMICEs in a large ! 9 number of species, the lack of functional studies to identify MICE single gene function and the poor sequence similarities with other bacterial systems raised two issues. The first relates to the criteria used for defining MICE during genome analyses.

For instance, ICEH of M. hyopneumoniae has been included in Figure 2 because it has been described as such but with only 12 genes ICEH stands apart [START_REF] Pinto | Molecular analysis of an integrative conjugative element, ICEH, present in the chromosome of different strains of Mycoplasma hyopneumoniae[END_REF]. ICEH displays a minimal MICE backbone in which CDS22 appears to be disrupted and was shown to excise from its host chromosome. Since CDS22 encodes the DDE recombinase responsible for MICE excision this data is puzzling.

The second question thus relates to vMICEs possibly still maintaining functions that may be activated or complemented under certain circumstances. In this review, MICEs were designated as such when displaying the minimal backbone (as defined above) within a locus of ca 30 kbps (see Figure 2) and/or there were some experimental evidences for their functional activity.

MICEs were mainly identified in the Hominis and the Spiroplasma phylogentic groups (Figure 1 [START_REF] Saillard | The abundant extrachromosomal DNA content of the Spiroplasma citri GII3-3X genome[END_REF]) and by the chromosome [START_REF] Carle | Partial chromosome sequence of Spiroplasma citri reveals extensive viral invasion and important g e n e d e c a y. A p p l E n v i r o n M i c r o b i o l[END_REF].

Interestingly, the Mycoides clade is composed of mycoplasma species infecting ruminants, with some sharing the same in-host niches with M. agalactiae or M. bovis. Based on their gene content, organization and sequences, MICEs found in ruminant species can be divided in two types that match with their phylogenetic position: the hominis and the spiroplasma types, with ICEA of strain 5632

and ICEM of strain GM12 as respective prototypes. This subdivision is particularly appropriate for the CDS22 transposase that is highly conserved within each MICE type but not in between types.

Small plasmids (ca. 2kbps) were found in mycoplasma of the Mycoides clade [START_REF] Breton | Distribution and diversity of mycoplasma plasmids: lessons from cryptic genetic elements[END_REF] together with MICEs raising the question of their interaction. In M. mycoides subsp. capri, an identical short non-coding region (ca. 150 nt) is present on both MICEs and plasmids [START_REF] Thiaucourt | Mycoplasma mycoides, from "mycoides Small C o l o n y " t o " capri" . A m i c r o e v o l u t i o n a r y perspective[END_REF], offering a means for recombination [START_REF] Wozniak | Integrative and conjugative elements: mosaic mobile genetic elements enabling dynamic lateral gene flow[END_REF]. Such co-integrates have yet to be isolated experimentally but they could provide small plasmids with MICE conjugative properties for their horizontal dissemination. In contrast, no plasmid was found in ruminant mycoplasma species of the Hominis phylogenetic group.

MICEs abundance in the two phylogenetic groups, their conjugative properties point towards these mobile elements as the main contributors, contrary to plasmids, of the massive HGT previously detected by computational analyses between M. agalactiae (Hominis group) and members of the Mycoides cluster (Spiroplasma group) (Sirand-Pugnet et al. 2007b).

MICE horizontal transmission

MICE propagation is secured by passive, vertical transmission during chromosomal replication and cell division. Functional studies performed with M. agalactiae have shown that ICEA is also able to self-disseminate horizontally via conjugation, a cellto-cell process occurring at a low frequency (ca 10 -7 transconjugants/donor strain) (Dordet-Frisoni et al. 2013b). Circumstances triggering this rare event are not known but we suspect that, as shown for other bacteria, chromosomal ICE genes are most often silent (see also below).

For cell-to-cell transfer, MICEs must first free themselves from the host chromosome. In M. agalactiae, this step involves short inverted repeats (IR, GGAA/TTCCC) located at the left and right end of the ICE which are then juxtaposed in the circular, extrachromosomal form after ICE excision. Free, circulate intermediates as well as empty chromosomal sites have been detected by PCR in several mycoplasmas species bearing ICEs [START_REF] Calcutt | Molecular genetic analysis of ICEF, an integrative conjugal element that is present as a repetitive sequence in the chromosome of Mycoplasma fermentans PG18[END_REF][START_REF] Vasconcelos | Swine and poultry pathogens: the complete genome sequences of two strains of Mycoplasma hyopneumoniae and a strain of Mycoplasma synoviae[END_REF] for some bacteria hosting mobile elements associated with DDE transposases [START_REF] Brochet | Atypical association of DDE transposition with conjugation specifies a new family of mobile elements[END_REF] but has yet not been detected in mycoplasmas. The two streptococcal ICEs, TnGBS1 and TnGBS2, also encode DDE recombinase and have no specific target site but preferentially insert upstream of promoter sequences [START_REF] Brochet | Atypical association of DDE transposition with conjugation specifies a new family of mobile elements[END_REF]. Overall, this contrast with the majority of bacterial ICEs described so far that use tyrosine recombinase, which one mediates site-specific recombination and often targets a specific attachment site (att) in the bacterial chromosome [START_REF] Johnson | Integrative and Conjugative Elements (ICEs): What they do and how they work[END_REF]. Altogether, experimental evidences have shown that ICEA is self-transmissible across cells and encode elements involved in its chromosomal excision, integration and in conjugation.

As mentioned earlier, the DDE recombinase, CDS22, is highly conserved within MICE types, the hominis-or the spiroplasma-type, but not in between types. IRs targeted by the recombinase during excision-integration are identical within MICE types but differ in between types, most likely reflecting a tight, specific interaction. Whether the DDE recombinases encoded by cds22 of the hominis-type are able to act on MICE of the spiroplasma type and vice et versa is not known, but it is interesting to speculate that the two ICE types may differ in their fine excision mechanism. Junctions of free circular MICEs (cMICEs) of the hominis type displayed a conserved sequence while those of the spiroplasma type only shared the same IR.

MICE conjugative machinery

MICEs transfer to a recipient cell via a conjugative process most likely as a single stranded DNA molecule with the help of SSB proteins. In mating conditions, ICE-negative strains of M. agalactiae are unable to undergo HGT unless they have received an ICEA. Thus, ICEA provides the recipient cell with the ability to conjugate, including the ability to exchange chromosomal DNA independently from ICE transfer (see below).

In bacteria, conjugation relies on a type IV secretion system which is expected to be minimal in the wallless mycoplasma cells. In their early work on chromosomal gene transfer in S. citri, Barroso and Labarère [START_REF] Barroso | Chromosomal gene transfer in Spiroplasma citri[END_REF] proposed that HGT might arise through fusion of donor and receptor cell membranes. Later, pili-like appendages were detected on the cell surface of S. kunkelii that connected two cells [START_REF] Ozbek | Infection and replication sites of Spiroplasma kunkelii (Class: Mollicutes) in midgut and Malpighian tubules of the leafhopper Dalbulus maidis[END_REF], suggesting that even simplified cells can produce sophisticated structures. Transmission or scanning microscopy of M. agalactiae strains, with or without ICE, in mating conditions or not, did not reveal any particular structure such as a pilus (unpublished data). The mycoplasma cells-membranes were however in intimate contact, with some structures resembling the nanotubes described by Dubey and Ben-Yehuda when studying neighboring Bacillus subtilis and Staphylococcus aureus or B. subtilis and Escherichia coli cells (Dubey and Ben-Yehuda 2011).

While membrane fusion during conjugation cannot be formally ruled out, several arguments point towards the synthesis of a conjugative channel between donor and recipient cells, as for other bacteria. In ICEA, candidates for the conjugative channel formation include seven gene products with transmembrane domains, namely CDS5, 7, 13, 15, 16, 17 and 19 (Figure 3). One additional surface lipoprotein, CDS14 is required for both ICE and chromosomal transfer: inactivation of cds14 abrogates conjugation, an event which is restored ! 11

upon complementation by the wild-type cds14 (Dordet-Frisoni et al. 2013b). CDS14 was confirmed experimentally to be expressed at the cell surface and was among the few ICE proteins to be detected by proteomics in standard laboratory conditions before mating (personal data). Interestingly, CDS14 does not have to be expressed by the ICE-donor cell as ICE transfer were documented in between an ICE-positive, donor strain deficient in CDS14 and an ICE-negative recipient strain in which wild-type CDS14 has been introduced. Based on these results, our current hypothesis is that CDS14 contributes to initiating the first conjugative step and is involved in cell-to-cell contact.

MICEs prevalence among mycoplasmas

The The Hominis phylogenetic group also contains species so far deprived of MICE such as the avian pathogen M. synoviae or the mice pathogen M. pulmonis for which only a few sequenced genomes are available in the databases. Finally, no MICE has been detected in the Acholeplasma phylogenetic group.

MICEs also vary in their copy number per hostchromosome, with a single strain having multiple copies of the same ICE, or multiple copies of two different ICEs. This is illustrated by the M. fermentans strain M64 which harbours three copies of ICEF-III and three copies of ICE-II [START_REF] Shu | Complexity of the Mycoplasma fermentans M64 genome and metabolic essentiality and diversity among mycoplasmas[END_REF]. MICE multiple occurrences complicate their proper identification in mycoplasma genome by providing repeated sequences that hampers correct assembly. Therefore, detailed MICE analyses in draft genomes should be taken with care and further completed by targeted, sequencing strategies. Overall, MICE strain-to-strain variability correlates with their highly dynamic nature and in turn poses the question of their fitness cost on the minimal mycoplasma cell or of their potential benefit, especially for those having multiple copies of similar elements.

MICE evolutionary history and trafficking

DDE recombinases (CDS22) are key elements of MICE mobility (Dordet-Frisoni et al. 2013b). A cascade of iterated Blast searches using the TnGBS2 transposase of streptococci as primary sequence revealed that MICE DDE recombinases constitute a new family of the Mutator and Mutatorlike superfamily in prokaryotes [START_REF] Guérillot | The diversity of prokaryotic DDE transposases of the mutator superfamily, insertion specificity, and association with conjugation machineries[END_REF]. Phylogenetic reconstructions further showed that they form a homogeneous, distinct clade, pMULT5 and are related to IS256, a widely spread insertion sequence [START_REF] Guérillot | The diversity of prokaryotic DDE transposases of the mutator superfamily, insertion specificity, and association with conjugation machineries[END_REF]). IS256r e l a t e d e l e m e n t s m o s t l i k e l y c i r c u l a t e i n mycoplasmas and have been found in the MICEbearing M. fermentans (ISMf1) [START_REF] Calcutt | Molecular genetic analysis of ICEF, an integrative conjugal element that is present as a repetitive sequence in the chromosome of Mycoplasma fermentans PG18[END_REF].

As expected, phylogenetic trees using cds22 and CDS5 sequences from predicted functional MICEs and from MICE vestiges indicate that in addition to vertical inheritance, MICEs are circulating across mycoplasma species and phylum.

The high prevalence of ICEs or vICEs in some mycoplasma species is largely due to their vertical chromosomal inheritance. This is illustrated within the Hominis group in which several species with distinct ecological niche harbor very similar MICEs (or MICE elements such as CDS5 or CDS17) as a result of vertical transmission from a common ancestor. These species include: M. fermentans, the human mycoplasma [START_REF] Calcutt | Molecular genetic analysis of ICEF, an integrative conjugal element that is present as a repetitive sequence in the chromosome of Mycoplasma fermentans PG18[END_REF], M. hyopneumoniae, a swine pathogen [START_REF] Minion | T h e g e n o m e s e q u e n c e o f M y c o p l a s m a hyopneumoniae strain 232, the agent of swine mycoplasmosis[END_REF][START_REF] Vasconcelos | Swine and poultry pathogens: the complete genome sequences of two strains of Mycoplasma hyopneumoniae and a strain of Mycoplasma synoviae[END_REF][START_REF] Liu | Complete genome sequence of Mycoplasma hyopneumoniae strain 168[END_REF], and several ruminant species such as M. bovis [START_REF] Wise | Complete genome sequence of Mycoplasma bovis type strain PG45 (ATCC 25523)[END_REF]) and M. agalactiae [START_REF] Marenda | A new integrative conjugative element occurs in Mycoplasma agalactiae as chromosomal and free circular forms[END_REF][START_REF] Nouvel | Comparative genomic and proteomic analyses of two Mycoplasma agalactiae strains: clues to the macro-and micro-events that are shaping mycoplasma diversity[END_REF] Finally, several reports have also suggested the natural occurrence of the TetM determinant in association with the Tn916 (see above). More recently, sequencing data have revealed the p r e s e n c e o f t r u n c a t e d T n 9 1 6 within an uncharacterized 25-Kbp transposon which is integrated into the rumA gene as for several closely related conjugative transposons found in streptococci [START_REF] Calcutt | An excisioncompetent and exogenous mosaic transposon harbors the tetM gene in multiple Mycoplasma hominis lineages[END_REF]. Interestingly, the 36.2% GC content of this transposon contrasted with the 27.1% of the core M. hominis genome. Whether this large mosaic element has retained competency for horizontal transfer is not known but its excision and circularization in M. hominis were supported by experimental evidences [START_REF] Calcutt | An excisioncompetent and exogenous mosaic transposon harbors the tetM gene in multiple Mycoplasma hominis lineages[END_REF].

Chromosomal transfer (CT) among mycoplasmas

In 2007, the first description of large-scale HGT among mycoplasmas sharing the same ecological niche marked a turn and challenged the generally accepted evolutionary scenario in which gene loss is the main driving force of mycoplasma evolution (Sirand-Pugnet et al. 2007b). In this study, comparative genomics together with phylogenetic tree reconstructions revealed that approximately 18% of M. agalactiae genome had undergone HGT with members of the phylogenetically distinct Mycoides cluster. No bias in the GC composition of the transferred genes could be observed that would distinguish them from their ancestors. Although base composition is often considered as a poor indicator of HGT [START_REF] Koski | Codon bias and base composition are poor indicators of horizontally transferred genes[END_REF], one possible explanation for this observation is that the acquired sequences have quickly adjusted to their new genome pattern. In M. agalactiae, HGT events often involved chromosomal genes clusters containing 2 to 12 elements with half of them displaying the same organization as in the Mycoides cluster genomes (Sirand-Pugnet et al. 2007b). The majority of these genes encodes for products with unknown function (40%) and/or with membrane-associated motifs (30%) while 15% have started to decay and were annotated as pseudogenes. Interestingly, M. agalactiae transferred genes were distributed all over the chromosome, a scattering that reflected multiple HGT events and/or extensive shuffling via intrachromosomal recombination events of the alien genes after integration.

With the concomitant discovery of MICEs in both M. agalactiae and Mycoides cluster genomes, conjugation quickly emerged as a possible mechanism for DNA transfer in mycoplasmas, with the hypothetical scenario in which MICEs could take along its host flanking regions. The conjugal transfer of marked MICEs was experimentally demonstrated in M. agalactiae from ICEA-positive to ICEAnegative cells (see above) but surprisingly no DNA of the donor chromosome was directly associated to MICE horizontal dissemination (Dordet-Frisoni et al. 2013b) (Figure 4). Indeed, observing and documenting the chromosomal transfer (CT) in mycoplasmas required a particular strategy: mating experiments were conducted with mycoplasma cells having the antibiotic selective markers inserted in different parts of the chromosome, outside of ICEA for ICEA-positive cells [START_REF] Dordet-Frisoni | Chromosomal transfers in mycoplasmas: when minimal genomes go mobile[END_REF]. Large-scale chromosomal DNA exchanges were then documented via conjugation but this event was observed from ICEA-negative to ICEA-positive strains (Figure 4). Again, no ICEA sequence was d i r e c t l y a s s o c i a t e d w i t h t h e t r a n s f e r r e d chromosomal sequences. CTs resulted in replacing large portions of the recipient mycoplasma genome by the donor counterparts via homologous recombination. By combining classical mating experiments with high-throughput next-generation sequencing, the transfer of nearly every position of the mycoplasma chromosome was detected. CTs were also experimentally achieved in between the two related species, M. agalactiae and M. bovis [START_REF] Dordet-Frisoni | Chromosomal transfers in mycoplasmas: when minimal genomes go mobile[END_REF]. HGT in between members of the Mycoides clade and the Hominis phylogenetic group remains to be documented in vitro but this is still facing the technical challenge of ! 13

finding laboratory conditions that can support their concomitant growth.

Conjugal transfer of chromosomal DNA is an old theme in bacterial evolution and is usually tightly associated with conjugative elements integrated into the chromosome, such as conjugative plasmids or ICEs, that provides the origin of transfer (oriT). In such system, both the transferred chromosomal segments and the conjugative element initially resided in the same donor cell, a situation contrasting with that described for M. agalactiae. As well, mobilization in trans of chromosomal region carrying cryptic oriT (IME) has also been described in several bacteria [START_REF] Daccord | Integrating conjugative elements of the SXT/ R391 family trigger the excision and drive the mobilization of a new class of Vibrio genomic islands[END_REF][START_REF] Bellanger | Conjugative and mobilizable genomic islands in bacteria: evolution and diversity[END_REF] but again this event is initiated in the same cell. Recently, an IS6-type element carried by a conjugative replicon in Yersinia pseudotuberculosis [START_REF] Lesic | A natural system of chromosome transfer in Yersinia pseudotuberculosis[END_REF]) was shown to drive the horizontal transfer of any large block of plasmid or chromosomal DNA but so far M. agalactiae or M. bovis are deprived of plasmid [START_REF] Breton | Distribution and diversity of mycoplasma plasmids: lessons from cryptic genetic elements[END_REF]). Bacterial conjugation is classically considered as being unidirectional [START_REF] Szpirer | Retrotransfer or gene capture: a feature of conjugative plasmids, with ecological and evolutionary significance[END_REF][START_REF] Bellanger | Site-specific accretion of an integrative conjugative element together with a related genomic island leads to cis mobilization and gene capture[END_REF]), but capture of conjugative elements or other cellular DNA by the so called donor has been occasionally reported. Yet, this is a two-steps model requiring first the transfer of the conjugative plasmid o r i n t e g r a t i v e e l e m e n t i n t o t h e r e c i p i e n t chromosomal DNA that is subsequently retrotransferred back into the donor. This model is appealing because ICEA integration occurs randomly and could theoretically retro-transferred equally any locus using its oriT. Yet it faces several ! 14 inconsistencies such as the observed frequency of chromosomal transfer being much higher than expected in such model. Rather, accumulating evidences point towards a mechanism resembling that described in Mycobacterium smegmatis. In this bacterium, a single recipient chromosome is able to acquire multiple, large unlinked segments of donor DNA [START_REF] Gray | Distributive conjugal transfer in mycobacteria generates progeny with meiotic-like genome-wide ! 18 mosaicism, allowing mapping of a mating identity locus[END_REF][START_REF] Gray | Intercellular communication and conjugation are mediated by ESX secretion systems in mycobacteria[END_REF][START_REF] Derbyshire | Distributive conjugal transfer: New insights into horizontal gene transfer and genetic exchange in Mycobacteria[END_REF], resulting in mosaic genomes. There is still much to be discovered about this process designated as DCT (distributive chromosomal transfer) but it is associated with the occurrence in the mycobacterium genome of multiple cis-acting sequences termed "bom" regions (basis of mobilization) that initiate transfers. Whether mycoplasmas also possess bom-like regions is not known and the mechanism underlying chromosomal transfer in these minimal bacteria has still to be fully understood. In our hands, two MICE-negative mycoplasma cells are unable to conjugate and no transconjugant is obtained unless the mycoplasma membranes are artificially fused by polyethylene glycol (PEG) (Dordet-Frisoni et al. 2013b[START_REF] Marenda | Genomic mosaics[END_REF]. Thus, our current working model is that CTs in mycoplasma require the presence of MICE in either one of the partners to form the conjugative bridge (Figure 4).

Overall, these finding challenged the classical mechanisms proposed for other bacteria in which CTs are driven by conjugative elements, bringing into the spotlight a new means for rapid mycoplasma innovation. They also radically change our current views concerning the evolution of mycoplasmas, with particularly far-reaching implications given that over 50 species are human or animal pathogens.

Extent of chromosomal horizontal transfer in mycoplasmas and biological impacts

Thanks to the tremendous advances made in NGS and in comparative genomic analyses, it is becoming now clear that chromosomal HGTs have played a major role in mycoplasma evolution and that this phenomenon is not restricted to ruminant mycoplasmas (see above).

HGT as a means for rapid adaptation

For instance, HGTs have been described in between the phylogenetic distinct species M. hominis and U. parvum, two colonizers of the human urogenital tract [START_REF] Pereyre | Life on arginine for Mycoplasma hominis: clues from its minimal genome and comparison with other human urogenital mycoplasmas[END_REF]). These events involved several clusters encoding restriction m o d i f i c a t i o n s y s t e m s , t r a n s p o s a s e s a n d hypothetical proteins, several of which display motifs associated with surface or membrane exposure and may play a role in the interaction with the host [START_REF] Pereyre | Life on arginine for Mycoplasma hominis: clues from its minimal genome and comparison with other human urogenital mycoplasmas[END_REF]. In silico genome comparative analyses combined to phylogenetic reconstructions also resulted in detecting HGT in between M. galisepticum and M. synoviae, two poultry pathogens belonging to two distinct phylogenetic groups (Sirand-Pugnet et al. 2007b).

Again, these events involved several loci predominantly encoding surface proteins and putative transposases [START_REF] Noormohammadi | Multigene families encoding the major hemagglutinins in phylogenetically distinct mycoplasmas[END_REF][START_REF] Wang | Limitations of compositional approach to identifying horizontally transferred genes[END_REF][START_REF] Vasconcelos | Swine and poultry pathogens: the complete genome sequences of two strains of Mycoplasma hyopneumoniae and a strain of Mycoplasma synoviae[END_REF]Sirand-Pugnet et al. 2007b).

In absence of a cell-wall, the mycoplasma membrane constitutes the primary interface with the host environment, mediating all types of direct and indirect interactions. It is therefore not surprising that transferred-genes, which were fixed over time, encode proteins with membrane associated motif and most likely are involved in mycoplasma-host interactions. Sharing genetic resources via horizontal gene transfer may provide mycoplasmas with a means for adapting to new niches or to new hosts while avoiding irreversible genome erosion.

HGT between Ureaplasma species questioned serotyping

Extensive HGTs have also been demonstrated in between human Ureaplasma species and serovars. In a study including over a 1000 clinical isolates of ureaplasmas (U. parvum and U. urealyticum), Li Xiao and co-workers discovered that 40% of the isolates were genetic mosaics, apparently arising from the recombination of multiple serovars (Xiao et al. 2011). Combined to the extensive comparative genomic analysis performed by [START_REF] Paralanov | Comparative genome analysis of 19 Ureaplasma urealyticum and Ureaplasma parvum strains[END_REF], their data supported the view that ureaplasmas exist as complex, highly heterogeneous populations rather than as stable serovars, due to HGT naturally occurring in these species. For clinicians, these data explain the extensive crossreactivity observed during serotyping and the lack of consistent association of given serotypes with disease, further implying that pathogenicity of ureaplasmas is unlikely to be associated with individual serovars, as it was speculated for decades (Xiao et al. 2011). Several isolates displayed in their genomes genes often associated to horizontal gene transfer such as traG/D homologs, Tn916 sequences, relaxases/mobilization proteins or homologs to phage integrases [START_REF] Paralanov | Comparative genome analysis of 19 Ureaplasma urealyticum and Ureaplasma parvum strains[END_REF]), but complete MICE have yet to be identified.

! 15

HGT in Spiroplasma and the emergence of new phenoytpes As regards to mycoplasmas infecting plants and insects, comparative and evolutionary genomics studies on Spiroplasma clades that included several species such as S. citri, S. chrysopicola, S. mirum and S. apis, revealed that the patterns of HGT varied widely [START_REF] Lo | Winding paths to simplicity: genome evolution in facultative insect symbionts[END_REF]. HGT is highly abundant in the most recent common ancestor of the Mirum clade, with an estimation of 7% of the S. eriocheiris protein-encoding genes coming from HGT [START_REF] Lo | Found and lost: The fates of horizontally acquired genes in arthropod-symbiotic Spiroplasma[END_REF]. Interestingly, most of these h a v e b e e n l o s t i n t h e s i s t e r s p e c i e s S. atrichopogonis, through accumulation of deletions. Due to the strong mutational bias towards deletions commonly observed in bacterial genomes (Kuo and Ochman 2009a) and the positive selection for the removal of pseudogenes (Kuo and Ochman 2010), losses of newly acquired genes is a common scenario unless these genes contribute to the fitness of their new bacterial host (Kuo and Ochman 2009b). The long-term survival of horizontally acquired genes in S. eriocheiris, a species which has experienced a host switch from terrestrial insects to aquatic crustaceans [START_REF] Bi | Phylogenetic analysis of Spiroplasmas from three freshwater crustaceans (Eriocheir sinensis, Procambarus clarkia and Penaeus vannamei) in China[END_REF][START_REF] Wang | Spiroplasma eriocheiris sp. nov., associated with mortality in the Chinese mitten crab, Eriocheir sinensis[END_REF], suggests that some of the novel acquired genes may be involved in the adaptation of a new ecological niche.

Besides the colonization of new niches, HGT could play an important role in the emergence of new strains or new phenotypes including for pathogenic species changes in virulence. This is illustrated in mosquito-associated species of the Apis clade (Lo et al. 2013b;[START_REF] Chang | Molecular evolution of the substrate utilization strategies and putative virulence factors in mosquito-associated Spiroplasma species[END_REF]) in which HGT is not as prevalent as for the Mirum or the Citri clades but may have had important consequences. In this clade, documented HGTs involve a virulence factor glpO [START_REF] Chang | Molecular evolution of the substrate utilization strategies and putative virulence factors in mosquito-associated Spiroplasma species[END_REF]) that is inferred as being present in the common ancestor of the Apis clade. This gene was then independently lost in the lineages leading to S. diminutum and S. sabaudiense, two non-pathogenic species for their mosquito host. In contrast, for two pathogenic species of mosquitos: S. culicicola which has maintained the vertically inherited copy and S. taiwanense which has acquired the entire gene cluster involved in glycerol metabolism (i.e., glpF-glpO-glpK) from a donor belonging to the Mycoides-Entomoplasmataceae clade. Notably, the horizontally acquired glpF-glpO-glpK in S. taiwanense appeared to have been integrated into the transcriptional control of their new host genome [START_REF] Lo | Horizontal acquisition and transcriptional integration of novel genes in mosquito-associated Spiroplasma[END_REF].

In spiroplasmas, mechanisms underlying HGT have yet to be elucidated but the presence of tra homologs (see above) together with experimental data [START_REF] Barroso | Chromosomal gene transfer in Spiroplasma citri[END_REF] tend to designate conjugation as one means. Yet a striking feature of the genomes of the Citri clade is the prevalence of sequences related to the plectrovirus genus that can account for ca. 20-30% of the chromosomes [START_REF] Carle | Partial chromosome sequence of Spiroplasma citri reveals extensive viral invasion and important g e n e d e c a y. A p p l E n v i r o n M i c r o b i o l[END_REF]Lo et al. 2013a). In addition to facilitating genome rearrangements, invasion of these viral sequences is associated with large fragments (i.e., >10 kb) of novel DNA at the viral insertion sites [START_REF] Ku | Complete genomes of two dipteran-associated spiroplasmas provided insights into the origin, dynamics, and impacts of viral invasion in spiroplasma[END_REF], suggesting that HGT in this clade may be mediated via transduction as well.

HGT occurs in mycoplasma sharing a same niche

A hallmark of mycoplasma DNA exchanges so far described is that donors and recipients colonize the same ecological niche. This is best illustrated by the trans-kingdom transfers recently documented in between the MRE by two independent studies concomitantly describing the occurrence of HGT in between MRE and their fungal host (Naito et globe with a striking lack of evidence of horizontal gene transfer (Xiao et al. 2015).

Finally, detecting Mycoplasmas HGT by in silico c o m p a r a t i v e g e n o m i c a n d p h y l o g e n e t i c reconstruction is often difficult in between closely phylogenetically related organisms or within the same species. Thus, our current view is most likely underestimating the role of these events.

Conclusions and future challenges

Despite having a minimal genome, several mycoplasmas have retained a form of sexual competence and are able to exchange significant amount of chromosomal material in addition to MGEs. This finding is radically changing the old paradigm of mycoplasma evolution being only driven by gene losses. Naito and Pawlowska [START_REF] Naito | The role of mobile genetic elements in evolutionary longevity of heritable endobacteria[END_REF] have recently proposed that this sexual competence is crucial for obligate intracellular mycoplasmas such as MREs to defy the effects of Muller's ratchet, a process in which accumulation and fixation of deleterious mutations drives to extinction small, asexual populations. Pathogenic intracellular or extracellular mycoplasma populations have to face various host responses and hostile environments during dissemination and colonization, going through continual bottlenecks.

Their limited genetic material and lack in DNA repair components make them particularly vulnerable to the deleterious effect of Muller's ratchet. HGT and the capacity to horizontally exchange any part of their genome may contribute to the maintenance of genomic information in mycoplasma subpopulations, providing them with a means to rescue injured genomes, restoring deleted or inactivated genes.

Except for genes that are lethal for the recipient mycoplasma cell, virtually any essential or accessory gene can be transferred. Thus, mycoplasma populations can be seen as a dynamic gene pool compensating genome erosion as well as clonality, allowing horizontal dissemination of genetic traits that may in turn result in the emergence of new strains, with new properties. While this may be beneficial to the survival of the minimal mycoplasma cells, such capacity may slow down the control of mycoplasma infectious diseases via the spreading genetic material associated to antibiotic resistance or virulence.

Understanding the mechanisms underlying and influencing mycoplasma HGT is an important issue but has just started to be explored. Since only conjugative transfers have been formally demonstrated so far, cell-to-cell contact constitutes the primary constraint for mycoplasma HGT to take place. Another is the occurrence of MICE in at least one partner to provide the conjugative pore (Figure 4). Indirect evidences also point towards additional HGT mechanisms such as transduction for spiroplasmas but the role of viruses in this phenomenon has yet to be addressed.

  Conjugative transfers are not restricted to the movement of plasmids or MGEs across bacterial

8 Figure 2 .

 82 Figure 2. Mycoplasma Integrative and Conjugative Element (MICE) backbone and structural organization of major MICE representatives. In MICE backbone, genes highly conserved across MICEs are positioned on the MICE (solid line) and are represented by colored filled arrows; conserved CDS that may be absent or truncated in particular MICEs are represented below the line. Inverted repeats (IR) are indicated by grey triangles. In classical MICE and degenerated vestigial MICE, highly conserved CDS are colored as in MICE backbone. Pseudogenes are represented by arrows filled with hatched color accordingly to MICE backbone. Genbank unique locus tag numbers of the first and the last MICE genes are indicated in blue below the MICE. Genes containing one or several transmembrane domains are indicated with an asterisk. Insertion sequences (IS) are represented by black boxes.

Figure 3 .

 3 Figure 3. ICE transfer in mycoplasmas. Overview of conjugative transfer in Gram-negative bacteria (Left panel) and proposed model for ICEA transfer in M. agalactiae (Right panel). Bacterial conjugation requires a close contact between the donor and recipient cells. This initial contact often involves complex structures, such as conjugative pili, in Gram-negative bacteria, but only a single surface lipoprotein (CDS14 product) has been so far identified as contributing to this intimate interaction in M. agalactiae. Upon induction, by specific cellular conditions or stochastic events, ICEs excise from the chromosome and forms a circular dsDNA molecule. In M. agalactiae, these two events are mediated by a DDE recombinase (CDS22 product). Following circularization, ICE-encoded proteins are actively expressed and assemble into the relaxosome complex at the origin of transfer (oriT), which process the circular ICE to generate a linear ssDNA-protein complex. ICEA circularization also induces the expression of the conjugative module (CDSs in gray) whose products assemble into the mating pore (conjugative channel), a simplified form of T4SS found in Gram-negative bacteria. Finally, the transfer of the single stranded ICE through the conjugative channel is facilitated by the TraG/VirD4 energetic component (CDS5 product) found at the inner side of the membrane. Once in the recipient strain, the ICE re-circularizes, becomes double stranded and integrates into the host chromosome. This schematic was adapted from Alvarez-Martinez and Christie (2009) and represents our current working model of conjugative transfer in mycoplasmas.

Figure 4 .

 4 Figure 4. Horizontal gene transfers in mycoplasmas: one ICE, but two mechanisms. Conjugative transfers in mycoplasmas are not restricted to ICE transmission, but can also involve the transfer of large chromosomal DNA fragments. During mating, DNA exchanges occur following two pathways including the horizontal transfer of the ICE (ICE transfer) from ICE-positive (ICE+) to ICE-negative (ICE-) cells and the transfer of chromosomal DNA (Chromosomal exchange) occurring in the opposite direction to the ICE movement. While mechanisms underlying ICE transfer in mycoplasmas share some similarity with the classical pathway (see Figure 3), chromosomal exchanges differ from the classical oriT-based conjugation. While taking advantage of the ICE conjugative machinery, chromosomal exchanges do not rely on the integration of ICE sequences in the transferred chromosomal sequences, but result in DNA swapping of large portions of the recipient mycoplasma genome by the donor counterparts via homologous recombination.

Table 1 .

 1 Taxonomy of genera included in the Mollicutes class.

				Number
	Order	Family	Genus	of species	Host
				(subspecies)
	Mycoplasmatales	Mycoplasmataceae	Mycoplasma	119 (4)	Animal, Human
			"Ca. Mycoplasma"	13 (2)
			Ureaplasma	7	Animal, Human
			Eperythrozoon a	2	Animal
	Haloplasmatales	Haloplasmataceae	Haloplasma	1	Sea
	Entomoplasmatales	Entomoplasmataceae	Entomoplasma	6	Insect, Plant
			Mesoplasma	12	Insect, Plants
		Spiroplasmataceae	Spiroplasma	36	Insect, Plants
	Acholeplasmatales	Acholeplasmataceae	Acholeplasma	15	Animal, Plant
			"Ca. Phytoplasma" b	38	Insect, Plant
	Anaeroplasmatales	Anaeroplasmataceae	Anaeroplasma	4	Bovine, Ovine
			Asteroleplasma	1	Bovine, Ovine

(from LPSN http://www.bacterio.net/mollicutes.html,

[START_REF] Tully | Revised taxonomy of the class Mollicutes: proposed elevation of a monophyletic cluster of arthropodassociated mollicutes to ordinal rank (Entomoplasmatalesord. nov.), with Provision for familial rank to separate species with nonhelical morphology (Entomoplasmataceaefam. nov.) from helical species (Spiroplasmataceae), and emended descriptions of the order Mycoplasmatales, family Mycoplasmataceae[END_REF]

; Johansson and Petterson 2002) a Most species of the genus Eperythrozoon have been transferred to the genus Mycoplasma except E. coccoides and E. parvum according to Rules of the Bacteriological Code, Uilenberg et al. IJSB 2006 proposed that the validly published names Eperythrozoon and Mycoplasma continue to be used as separate genera possibly in the same Mycoplasmataceae family b Genus of uncertain taxonomy composed by in vitro uncultivable species. Phylogenetically near of acholeplasmas, and provisory classed in the Acholeplasmataceae family.

  , CDS22, which gene is located at one end of the element(Dordet-Frisoni et al. 2013b;[START_REF] Guérillot | The diversity of prokaryotic DDE transposases of the mutator superfamily, insertion specificity, and association with conjugation machineries[END_REF]. DDE recombinases are typically associated with IS, transposon or phage and do not usually target specific site for integration[START_REF] Brochet | Atypical association of DDE transposition with conjugation specifies a new family of mobile elements[END_REF]. Indeed, ICEA integration into the recipient chromosome is totally random, occurring at different loci, mostly in putative coding regions. This was demonstrated by direct sequencing of host-chromosomal regions that flanked the integrated ICE in a series of transconjugants derived from a single pair. As well, random chromosomal insertions of a mini-ICE construct that carried cds22 and a selection maker flanked by ICEA inverted repeats were obtained upon transformation of a M. agalactiae ICE-negative strain(Dordet-Frisoni et al. 2013b). Integration of MICE circular forms results in the duplication of 8 nt of the host chromosome that end up on each side of the element as direct repeats, an event leaving no trace after ICE excision[START_REF] Calcutt | Molecular genetic analysis of ICEF, an integrative conjugal element that is present as a repetitive sequence in the chromosome of Mycoplasma fermentans PG18[END_REF][START_REF] Marenda | A new integrative conjugative element occurs in Mycoplasma agalactiae as chromosomal and free circular forms[END_REF] Dordet-Frisoni et al. 2013b). In M. agalactiae, close examination of the ICEA integration sites revealed no polymorphism indicative of the presence of the donor chromosome other than that encoded by the ICE suggesting a clean cut and paste mechanism. Indeed, extrachromosomal circular forms of ICEA as well as chromosomal empty sites were detected in all ICEA transconjugants tested so far. Transient replication of ICE-circulate intermediates has been described

;[START_REF] Marenda | A new integrative conjugative element occurs in Mycoplasma agalactiae as chromosomal and free circular forms[END_REF][START_REF] Pinto | Molecular analysis of an integrative conjugative element, ICEH, present in the chromosome of different strains of Mycoplasma hyopneumoniae[END_REF][START_REF] Tardy | Integrative conjugative elements are widespread in field isolates of Mycoplasma species pathogenic for ruminants[END_REF]

. MICE excision and subsequent integration in the new recipient chromosome is driven by a DDE ! 10 recombinase

  While sharing the same ecological niche is an obvious facilitator of HGT, other environmental or genetic factors such ICEs are required. For instance, M. genitalium also colonizes the human urogenital but there is no evidence of HGT with the M. hominis or U. parvum co-resident species. Like M. genitalium, M. pneumoniae appears to be deprived of MICEs and a recent comparative genomic analysis of strains isolated between 1940 to 2009 indicate that its genome is extraordinarily stable over time and geographic distance across the

	al.
	2015; Torres-Cortés et al. 2015) although data were
	obtained with MRE populations associated with
	different AMF hosts. Briefly, MRE genomes were
	shown to harbor genes horizontally acquired from
	AMF that encoded a large repertoire of proteins with
	eukaryotic domains. For instance, these trans-
	kingdom transfers involved genes encoding small
	ubiquitin-like modifier (SUMO) proteases specific to
	eukaryotic SUMOylation systems. Although
	experimental data are needed, fungal SUMO
	proteases might play an important role for MRE by
	providing them with a means for manipulating the
	fungal host cytoplasmic environment in order to
	increase MRE fitness. Altogether, the large number
	of acquired genes with eukaryotic domains suggests
	the MRE high degree of adaptation to their fungal
	host (Kuo 2015; Naito et al. 2015; Torres-Cortés et
	al. 2015).

! 16

  Finally, in mycoplasmas, viable conjugal transfers are detected at very low frequencies (10 -7 ) under laboratory conditions most likely because of MICE expression being shut down. MICE excision and subsequent horizontal transfers might be modulated by external factors such as physical stresses (i. e. temperature)[START_REF] Tardy | Integrative conjugative elements are widespread in field isolates of Mycoplasma species pathogenic for ruminants[END_REF], or by host-or MICEs-genetic factors. Our current hypothesis is that CTs are intimately linked to the MICE-encoded conjugal pore, thus deciphering the environmental and mycoplasma factors that can trigger or repress MICE expression is currently emerging as a primary question. Rev Microbiol 8:552-563. doi: 10.1038/ nrmicro2382 Xiao L, Paralanov V, Glass JI, et al (2011) Extensive horizontal gene transfer in ureaplasmas from humans questions the utility of serotyping for d i a g n o s t i c p u r p o s e s . J C l i n M i c r o b i o l 49:2818-2826. doi: 10.1128/JCM.00637-11 Xiao L, Ptacek T, Osborne JD, et al (2015) Comparative genome analysis of Mycoplasma pneumoniae. BMC Genomics 16:610. doi: 10.1186/s12864-015-1801-0 Xie J, Tiner B, Vilchez I, Mateos M (2011) Effect of the Drosophila endosymbiont Spiroplasma on parasitoid wasp development and on the reproductive fitness of wasp-attacked fly survivors. Evol Ecol 53:1065-1079. doi: 10.1007/ s10682-010-9453-7 Xie J, Vilchez I, Mateos M (2010) Spiroplasma bacteria enhance survival of Drosophila hydei attacked by the parasitic wasp Leptopilina heterotoma. PloS One 5:e12149. doi: 10.1371/ journal.pone.0012149.

! 22

Acknowledgements

Due to length constraints, many references could not be cited. Our work on the role of MGE and on chromosomal transfer was supported by the French National Research Agency, ANR, (MycXGene, ANR09MIE016 coordinated by CC), by the National Institute for research in Agriculture (INRA), and by the national veterinary school of Toulouse (ENVT). Funding to CHK was provided by Academia Sinica (Taipei, Taiwan).


Maurice Fadel
email: fadel@laplace.univ-tlse.fr

Desmana Tri

Rachmildha

Control of Photovoltaic System Connected Directly To The Grid by a 3-Phase BOOST Inverter

Keywords: Photovoltaic system, Solar energy, Conversion losses, Topology of conversion, Grid connected, Power electronics.  

For PV applications, many studies focus on maximum power extraction without evaluating losses in the conversion chain. As part of this research, it is proposed to evaluate a direct energy conversion solution from the panel to the network without intermediate stages or transformers. The structure is presented as a 3-phase boost inverter integrating the maximum power extraction function (MPPT) while ensuring control of the power fed into the grid. A precise assessment of the losses will be carried out and an evaluation on the different components will be compared. The operation will be studied in particular according to the power rating and the switching frequency.

INTRODUCTION

The grid-connected photovoltaic systems are currently being used more and more in the power system due to the fact that their price is becoming lower and competitive compared to other power plants [START_REF] Kjaer | A Review of Single-Phase Grid-Connected Inverters for Photovoltaic Modules[END_REF][START_REF]A Review of Grid-Tied Converter Topologies Used in Photovoltaic Systems[END_REF]. But since the energy produced is in the form of DC low voltage, it is important to convert the voltage to make it compatible with the distribution network (single-phase AC or 3-phase AC). The solutions to this problem oftenly involve the utilization of high frequency transformers or single stage or multi-stage Boost converters to avoid the use of low frequency transformers. Although high frequency transformers offer flexible range of output voltage, the design is considered quite complex and difficult. On the other hand, the use of additional stage of converters require more power switches as well as independent control blocks. This can lead to higher price for the converter. In this paper, the direct conversion from lower dc to higher ac voltage is proposed using so called Boost inverter for 3-phase system, using only 6 power switches [START_REF] Jain | Single-Stage Grid Connected Inverter Topology for Solar PV Systems With Maximum Power Point Tracking[END_REF][START_REF] Caceres | A Boost DC-AC Converter: Analysis, Design, and Experimentation[END_REF]. However, many publications focus on maximum energy recovery via MPPT, but few focus on the efficiency of the conversion chain, including the study of losses in the different components of the conversion topology. Whether for single-phase or three-phase structures, the use of a Boost converter mounted as an inverter becomes a worthwhile solution due to its small number of components [START_REF] Devesh | Modeling and designing of solar photovoltaic system with 3 phase grid connected inverter[END_REF], [START_REF] Arvind | Thakura Assessement of Single Stage Three Phase Boost Inverter for an Islanded Microgrid[END_REF]. In addition, the large presence of passive components requires a more detailed study of the total conversion losses. In this research, it is proposed to conduct a study on the different losses as a function of the working power and the switching frequency used. The parameters used in this circuit can be indicated in To ensure correct operation of the converter, the voltages across the capacitors will be controlled as written in the Eq.

(1) :

1 2 3
.sin( . )

.sin( . 2. / 3)

.sin( . 2. / 3)

cd c cd c cd c V V Vt V V Vt V V Vt         With 550 v dc V 
In order to control this converter, the following objectives must be taken into account, i.e. extract the maximum power at the photovoltaic panel [START_REF] Femia | Optimization of perturb and observe maximum power point tracking method[END_REF] and inject the power into the grid with sinusoidal currents in-phase with the grid voltages to have zero reactive power. Figure 2 shows the structure of the general control system of the inverter. The transfer of energy from the photovoltaic panel to the grid is accompanied by losses that can be classified into 2 categories, losses in passive components (inductances and capacitors) and losses in power switches (diode and transistors) [START_REF] Takao | Novel exact power loss design method for high output power density converter[END_REF]. Losses depend on several factors such as temperature, average current, current ripple and switching frequency. The objective of this study is to know the losses happen in the converter as the function of power rating and the switching frequency.

Power dissipation loss calculation

This section explains the simplified method of calculating energy dissipation for IGBT modules. It is used to calculate the power dissipation for different operating conditions with different IGBT modules. It is based on Fuji Electric's [START_REF] Fuji | [END_REF] rating. The IGBT module consists of several IGBT matrices and FW Diode matrices. The sum of the power losses of these matrices is equal to the total power loss of the module. The power loss can be classified as a working loss or a switching loss. A diagram of the power loss factors is shown in Figure 1 below. It shows the different losses in the transistor and diode that combine to provide the total losses. The operating power losses of the IGBT and FW Diode elements can be calculated using the output characteristics, and the switching losses can be calculated from the switching loss characteristics according to the collector current on the data sheet. Use these power loss calculations to design an appropriate cooling system to keep the junction temperature Tj below the maximum nominal value. The values of the switching voltage and switching loss at the standard junction temperature (Tj=150°C) are recommended for the calculation as follows

Thermal Module of PSIM

Thermal Module [START_REF] Powersim | PSIM User's Guide[END_REF] is the element to calculate the loss of power semiconductor devices in PSIM. Using thermal module, it is possible to simulate the loss of converter quickly and accurately based on the datasheets of diodes, IGBTs. The simulation schematic using thermal modules generates the values of semiconductor loss, conduction loss and switching loss [START_REF] Takao | Novel exact power loss design method for high output power density converter[END_REF]. A device database editor is provided to allow users to add new devices to the database and to manage the database easily and then can be used in the simulation for the loss calculation. The following illustrates the process of how a device in the database is used in the simulation and how the losses are calculated:

-The behavior model of the device is used in the simulation. The behavior model takes into account the static characteristics of the device (such as conduction voltage drop, on-state resistance, etc.), but not the dynamic characteristics (such as turn-on and turn-off transients). -Based on the voltage, current, and junction temperature values from the simulation, PSIM accesses the device database and calculates the conduction losses or switching losses. The static characteristics of the device are updated for the next simulation. Note that the loss calculation is only an approximation, and the accuracy of the results depends on the accuracy of the device data as well as the proper scaling of the results from the device test condition to the actual circuit operating conditions.

Losses in passive components

In order to calculate losses in passive components, it is necessary to consider real components, which also allows us to have more realistic simulations. In a real coil, the losses are translated by: -losses in the winding due to the resistance of the yarn that composes it, they are modelled by a series resistance (equivalent series resistance); -losses in the magnetic core: losses by hysteresis and eddy currents, they are modelled by a parallel resistance; -losses due to parasitic capacitance created between each turn, they depend on the winding method and are modelled by a parallel capacitance.

In order to estimate the ESR values for each coil examined in this study, series/parallel combinations of the 26µH [9] high-current coil to obtain the desired value and satisfy the nominal currents are used.

For the actual capacitor depicted in Fig. 4, the loss factors are:

-conduction losses, related to a leakage current in the insulation and represented by a parallel resistance called insulation resistance;

-dielectric hysteresis losses that cause the dielectric to heat up when subjected to an alternating electric field. They are modelled by a series resistance whose value depends on the frequency.

A series inductance (ESL) represents the parasitic inductances generated by the construction of the capacitor. The insulation resistance is usually very high (some MΩ), so conduction losses have been neglected in this study. The ESR and ESL of the capacitors used in the simulations were obtained in the datasheet [START_REF] Kemet | [END_REF] for electrolytic capacitors and in the datasheet [11] for film capacitors. For the output capacitors the following reference is used 1000VDC-440VAC.

IV. CONTROL BLOCKS AND SIMULATION RESULTS

The operation of the converter requires the control of several variables simultaneously. Here a cascade diagram using several loops is used, ensuring the control of the currents injected into the network. First, it is necessary to produce the current references which are determined from the reference power. The next step is to control the capacitor voltages and finally to control of the currents in the input inductances. These 3 loops must satisfy an adapted and coherent dynamics [START_REF] Moussa | A Single-Stage DC-AC Boost Topology and Control for Solar PV Systems supplying a PMSM[END_REF].

This control structure in Fig. 5 shows 3 loops. The fastest dynamic response is defined on the frequency scale by the bandwidth frequency ωbp1 located 1/2 decade from the switching frequency ωd. It concerns about the control of inductor currents IL1,2,3. The second loop deals with the adjustment of the voltages across the capacitors. The dynamics is defined by the frequency ωbp2 located at 1/2 decade lower than ωbp1. Finally, the third loop, the slowest, is characterized by a frequency ωbp3, and it is located 1/2 from the ωbp2. It is important to note that the frequency ωbp3 must be at 3 times higher than the frequency of grid. In this paper, if the chosen switching frequency is 10 kHz, than the input inductor current controller (represented by C3(s)) will have 2000 Hz of bandwidth, the capacity or voltage controller will have 400 Hz (represented by C2(s)), and the chosen bandwidth for the grid current controller is 200 Hz. Thus, the time constants for the controllers C3, C2 and C1 are 137.8 us, 689.12 us, and 1.4 ms respectively.

The control block along with the inverter circuit are simulated using PSIM. Figure 6 shows the result of the simulation of the inductor current, voltage across the capacitors, and the current injected to the grid, at 10kW power rating. Note that the current waveforms of the input inductors is non-sinusoidal and it is a normal characteristic in this type of converter.

The control block along with the inverter circuit are simulated using PSIM. Figure 6 shows the result of the simulation of the inductor current, voltage across the capacitors, and the current injected to the grid, at 10kW power rating.

Figure 6. Waveforms of currents and capacitor voltages for the power rating of 10 kW

M P  C P ref ,Q ref dq abc + - dq abc I d_ref ,I q_ref I d ,I q C 1 (s) V cd_ref , V cq_ref + - V C1,C2,C3 C 2 (s) + - I ref_L1,L2,L3 I L1,L2,L3 C 3 (s) α 1,2,3 V d ,V q V A,

B,C I A,B,C (Grid voltages) (Actual injected currents) (Actual capacitor voltages) (Actual inductor currents)

dq abc Figure 8. Distribution of losses according to power (kW) at 10 kHz switching frequency Note that the current waveforms of the input inductors is nonsinusoidal and it is a normal characteristic in this type of converter. Note also that, from Fig. 6, the capacitor voltages are biased with 550 V DC, measured from the boost inverter ground point. Nevertheless, the injected currents are sinusoidal without dc component as represented for phase a still in Fig. 6. The success of MPPT algorithm is also represented in Fig. 6, and the input power detected is around 10 kW (9 841 Watts).

V Cref _ 123 ω d ω bp1 ω bp2 ω bp3

V. STUDY OF THE LOSSES OF THE CONVERSION CHAIN

Here, the study of losses in this converter's structure as a function of the power rating which is varied between 2 and 25 kW is proposed. Losses are analyzed separately in accordance with the passive and active components. The study also examines the role of the switching frequency, which is varied between 5 and 25 kHz. The chosen power switch Powerex CM600DU-24F and the losses characteristic for each commutation to ON and OFF state of the device as the function of collector current, Ic, can be depicted in Fig. 7. The conduction losses occurred in each IGBT and its paralleled diode are also considered, and the voltage drop characteristic for each element is also recorded in the simulator. Beside the losses occurred in the power switches, the losses in passive components (inductors and capacitors) are also considered using the parasitic resistances as already mentioned in Table 1.

V.1 Study according to the power rating

The overall performance, for a switching frequency of 10 kHz, is presented in Table 2, where the distribution of losses by category is observed.

Table 2. Conversion efficiency

In general, the efficiency increases with power and the weak point of this structure is its efficiency at low powers. It is important to analyze the source of the losses and link them to the components, as shown in Fig. 8. Fig. 8 also shows that the majority of losses are attributed to active components with a contribution of at least 80%.

It should be noted that 60% relates to the switching losses and 20% to conduction losses. These results are inherent to the structure of the converters, which must maintain a DC component across the capacitors in order to impose sinusoidal voltages line to line.

V.2 Study according to the switching frequency Fig. 9 shows the evolution of the overall efficiency of the conversion chain as a function of the switching frequency for several power ratings. The curve shows that the efficiency becomes maximum around 10 kHz and then decreases inexorably. Fig. 9 also shows the distribution of losses in this context. This curve confirms the significant losses in the active components and the linear dependence on the switching frequency. In this research, a study on a single stage grid connected 3phase Boost inverter has been conducted. The results clearly show that the efficiency increases with the converted power in high proportions (from 68 to 93%). This type of converter will be more interesting as the power rating varies slightly as it is the case for use in tropical areas. Indeed, in tropical areas the irradiance is substantially constant throughout the day due to the positioning of the sun that does not leave the intertropical band. The total losses are dominated by the switching losses therefore it is subjected to the further research on how to deal with this type of losses. The use of low losses type of switch can be considered in this type of converter. It is also interesting to observe that the efficiency also varies with the switching frequency. In this design, it is observable that the efficiency is best at around 10 kHz.

2 . 3 -Figure 1 .

 231 Figure 1. Structure of 3-phase boost inverter connected to a grid

Figure 2 .

 2 Figure 2. Structure of control

Figure 3 .

 3 Figure 3. Model of a real coil Figure 4. Model of a real capacitor Figure 3 describes the detailed model of a real coil. The losses in parasitic capacitance become significant when the coil is injected with high frequencies. But in this study, only the losses in the series resistance are therefore taken into

Figure 5 .

 5 Figure 5. Control diagram with associated bandwidths

Figure 7 .

 7 Figure 7.a. Eon characteristic Figure 7.b. Eoff characteristic

Figure 9 .Figure 10 .

 910 Figure 9. Efficiency as a function of switching frequency for several power ratings

Table

ACKNOWLEDGMENT

The authors would like to thank the Ministry of Europe and Foreign Affairs for their support through the PHC NUSANTARA 2018 program. This support (Project no. 41190WM) made it possible to connect the Bandung ITB to the LAPLACE Laboratory to conduct research in the field of renewable energy.