Horizontal Gene Transfers in Mycoplasmas ( Mollicutes )

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 integrat ive 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 (Frost et al. 2005; Ochman et al. 2000). 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 (Halary et al. 2010; Kloesges et al. 2011). 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 (Guglielmini et al. 2011). 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 (Bellanger et al. 2014). 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 (Daccord et al. 2010). Conjugative transfers are not restricted to the movement of plasmids or MGEs across bacterial !3 Curr. Issues Mol. Biol. (2018) 29: 3-22. caister.com/cimb Horizontal Gene Transfers in Mycoplasmas


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 (Frost et al. 2005;Ochman et al. 2000).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 (Halary et al. 2010;Kloesges et al. 2011).
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 (Guglielmini et al. 2011).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 (Bellanger et al. 2014).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 (Daccord et al. 2010).cells but can also involve chromosomal DNA regions as initially observed by Lederberg andTatum in 1946 (Lederberg andTatum 1946).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 (Hochhut et al. 2000), mainly because of physical constraints applying on large molecules during transfer.More recently, chromosomal transfers have been described in Mycobacterium smegmatis (Gray et al. 2013;Derbyshire and Gray 2014) and in Mycoplasma agalactiae (Dordet-Frisoni et al. 2014) 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.

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 (McCutcheon and Moran 2011;Moran and Bennett 2014).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 (Gibson et al. 2010).

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 (Razin and Hayflick 2010;Citti and Blanchard 2013).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.

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;Pereyre et al. 2009).The concomitant detection of putative ICEs (Calcutt et al. 2002;Marenda et al. 2006)

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 (Saitou and Nei 1987).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 (Felsenstein 1992).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 (Felsenstein 1992).The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura et al. 2004) 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 (Kumar et al. 2016).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/).
With the advances in genome sequencing, several other MICEs have since been described which overall organization is relatively similar (Calcutt et al. 2002;Pinto et al. 2007;Nouvel et al. 2010;Wise et al. 2011;Thiaucourt et al. 2011;Shu et al. 2012) 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 (Pinto et al. 2007).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

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 (Barroso and Labarère 1988) 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 (Ozbek et al. 2003), 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 (Shu et al.

2012). 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.2015).

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 (Koski et al. 2001), 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 (Dordet-Frisoni et al. 2014).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 (Dordet-Frisoni et al. 2014).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 (Daccord et al. 2010;Bellanger et al. 2014) but again this event is initiated in the same cell.Recently, an IS6-type element carried by a conjugative replicon in Yersinia pseudotuberculosis (Lesic et al. 2012) 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 (Breton et al. 2012).Bacterial conjugation is classically considered as being unidirectional (Szpirer et al. 1999;Bellanger et al. 2011), 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 (Gray et al. 2013(Gray et al. , 2016;;Derbyshire and Gray 2014), 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, 2014).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 (Pereyre et al. 2009).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 (Pereyre et al. 2009).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).
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 Paralanov et al. (2012), 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 (Paralanov et al. 2012), 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 (Lo et al. 2016).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 (Lo et al. 2015).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 (Bi et al. 2008;Wang et al. 2011), 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;Chang et al. 2014) 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 (Chang et al. 2014) 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 (Lo and Kuo 2017).
In spiroplasmas, mechanisms underlying HGT have yet to be elucidated but the presence of tra homologs (see above) together with experimental data (Barroso and Labarère 1988)  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 .
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 .
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 .
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 .
Taxonomy of genera included in the Mollicutes class.
(from LPSN http://www.bacterio.net/mollicutes.html,Tully et al. 1993; 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.
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.mycoidesorM. capricolum capripneumoniae, along with Spiroplasma species(Gasparich et al. 2004) 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(Nunan et al.  2005)of the Citri clade for saltwater shrimps and S. eriocheiris(Wang et al. 2011)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(Tinsley and Majerus  2006).Interestingly, some Spiroplasma spp.have evolved as defensive mutualists providing their Drosophila hosts with a protection against nematodes (Jaenike et al. 2010) or parasitic wasps(Xie et al. 2010(Xie et al.  , 2011)).Finally, only one Spiroplasma species is known as a pathogen of vertebrates (Tully 1982) namely S. mirum, the suckling mouse cataract agent(Tully et al. 1976(Tully et al.  ).al. 2015;; Torres-Cortés et al. 2015).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 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.

Mycoplama Integrative Conjugative Elements (MICEs): a new family of self-transmissible element with conjugative properties
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.
et al. 2002;blast search retrieved the presence of CDS5 and CDS17 homologs in Ureaplasma of the Pneumoniae group such as in the U. diversum genome of strain ATCC 49782 (Marques et al. 2015, 2016, see also below) but not in Phytoplasma which include non-cultivable species.Within the Spiroplasma phylogenetic group, MICEs occur within the Mycoides clade (Figure 1) and display a global nucleotide identity ranging between 68 and 89%.These include ICEC and ICEM respectively found in M. capricolum subsp.capricolumand in M. mycoides subsp capri, (Sirand-Pugnet et al. 2007b; Thiaucourt et al. 2011).Putative homologs of traG and/or traE (also designated traE) have been found in several Spiroplasma genomes (Saillard et al. agalactiae (Hominis group) and members of the Mycoides cluster (Spiroplasma group) (Sirand-Pugnet et al. 2007b).severalmycoplasmasspecies bearing ICEs (Calcuttet al. 2002; Vasconcelos et al. 2005; Marenda et al.  2006; Pinto et al. 2007; Tardy et al. 2015).MICE excision and subsequent integration in the new recipient chromosome is driven by a DDE ! 10 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.
late discovery of MICEs is partly due to their variable occurrence within and among Mollicutes species.Hence, MICEs appear to be completely lacking in some species such as M. pneumoniae or M. genitalium of the Pneumoniae phylogenetic group, two human pathogens for which a high number of strains have already been sequenced.In contrast, the Hominis group is rich of mycoplasmas species bearing MICEs such as the human M.