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1.  Chromosomal Transfers in Mycoplasmas: When Minimal Genomes Go Mobile 
mBio  2014;5(6):e01958-14.
ABSTRACT
Horizontal gene transfer (HGT) is a main driving force of bacterial evolution and innovation. This phenomenon was long thought to be marginal in mycoplasmas, a large group of self-replicating bacteria characterized by minute genomes as a result of successive gene losses during evolution. Recent comparative genomic analyses challenged this paradigm, but the occurrence of chromosomal exchanges had never been formally addressed in mycoplasmas. Here, we demonstrated the conjugal transfer of large chromosomal regions within and among ruminant mycoplasma species, with the incorporation of the incoming DNA occurring by homologous recombination into the recipient chromosome. By combining classical mating experiments with high-throughput next-generation sequencing, we documented the transfer of almost every position of the mycoplasma chromosome. Mycoplasma conjugation relies on the occurrence of an integrative conjugative element (ICE) in at least one parent cell. While ICE propagates horizontally from ICE-positive to ICE-negative cells, chromosomal transfers (CTs) occurred in the opposite direction, from ICE-negative to ICE-positive cells, independently of ICE movement. These findings challenged the classical mechanisms proposed for other bacteria in which conjugative CTs are driven by conjugative elements, bringing into the spotlight a new means for rapid mycoplasma innovation. Overall, they 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.
IMPORTANCE
Horizontal gene transfers (HGT) shape bacterial genomes and are key contributors to microbial diversity and innovation. One main mechanism involves conjugation, a process that allows the simultaneous transfer of significant amounts of DNA upon cell-to-cell contact. Recognizing and deciphering conjugal mechanisms are thus essential in understanding the impact of gene flux on bacterial evolution. We addressed this issue in mycoplasmas, the smallest and simplest self-replicating bacteria. In these organisms, HGT was long thought to be marginal. We showed here that nearly every position of the Mycoplasma agalactiae chromosome could be transferred via conjugation, using an unconventional mechanism. The transfer involved DNA blocks containing up to 80 genes that were incorporated into the host chromosome by homologous recombination. These findings radically change our views concerning mycoplasma evolution and adaptation with particularly far-reaching implications given that over 50 species are human or animal pathogens.
doi:10.1128/mBio.01958-14
PMCID: PMC4251992  PMID: 25425234
2.  Predicting the Minimal Translation Apparatus: Lessons from the Reductive Evolution of Mollicutes 
PLoS Genetics  2014;10(5):e1004363.
Mollicutes is a class of parasitic bacteria that have evolved from a common Firmicutes ancestor mostly by massive genome reduction. With genomes under 1 Mbp in size, most Mollicutes species retain the capacity to replicate and grow autonomously. The major goal of this work was to identify the minimal set of proteins that can sustain ribosome biogenesis and translation of the genetic code in these bacteria. Using the experimentally validated genes from the model bacteria Escherichia coli and Bacillus subtilis as input, genes encoding proteins of the core translation machinery were predicted in 39 distinct Mollicutes species, 33 of which are culturable. The set of 260 input genes encodes proteins involved in ribosome biogenesis, tRNA maturation and aminoacylation, as well as proteins cofactors required for mRNA translation and RNA decay. A core set of 104 of these proteins is found in all species analyzed. Genes encoding proteins involved in post-translational modifications of ribosomal proteins and translation cofactors, post-transcriptional modifications of t+rRNA, in ribosome assembly and RNA degradation are the most frequently lost. As expected, genes coding for aminoacyl-tRNA synthetases, ribosomal proteins and initiation, elongation and termination factors are the most persistent (i.e. conserved in a majority of genomes). Enzymes introducing nucleotides modifications in the anticodon loop of tRNA, in helix 44 of 16S rRNA and in helices 69 and 80 of 23S rRNA, all essential for decoding and facilitating peptidyl transfer, are maintained in all species. Reconstruction of genome evolution in Mollicutes revealed that, beside many gene losses, occasional gains by horizontal gene transfer also occurred. This analysis not only showed that slightly different solutions for preserving a functional, albeit minimal, protein synthetizing machinery have emerged in these successive rounds of reductive evolution but also has broad implications in guiding the reconstruction of a minimal cell by synthetic biology approaches.
Author Summary
In all cells, proteins are synthesized from the message encoded by mRNA using complex machineries involving many proteins and RNAs. In this process, named translation, the ribosome plays a central role. The elements involved in both ribosome biogenesis and its function are extremely conserved in all organisms from the simplest bacteria to mammalian cells. Most of the 260 known proteins involved in translation have been identified and studied in the bacteria Escherichia coli and Bacillus subtilis, two common cellular models in biology. However, comparative genomics has shown that the translation protein set can be much smaller. This is true for bacteria belonging to the class Mollicutes that are characterized by reduced genomes and hence considered as models for minimal cells. Using homology inference approach and expert analyses, we identified the translation apparatus proteins for 39 of these organisms. Although striking variations were found from one group of species to another, some Mollicutes species require half as many proteins as E. coli or B. subtilis. This analysis allowed us to determine a set of proteins necessary for translation in Mollicutes and define the translation apparatus that would be required in a cellular chassis mimicking a minimal bacterial cell.
doi:10.1371/journal.pgen.1004363
PMCID: PMC4014445  PMID: 24809820
3.  An international collaborative study to determine the prevalence of contagious caprine pleuropneumonia by monoclonal antibody-based cELISA 
Background
Few serological tests are available for detecting antibodies against Mycoplasma capricolum subsp. capripneumoniae, the causal agent of contagious caprine pleuropneumonia (CCPP). The complement fixation test, the test prescribed for international trade purposes, uses a crude antigen that cross-reacts with all the other mycoplasma species of the “mycoides cluster” frequently infecting goat herds. The lack of a more specific test has been a real obstacle to the evaluation of the prevalence and economic impact of CCPP worldwide. A new competitive ELISA kit for CCPP, based on a previous blocking ELISA, was formatted at CIRAD and used to evaluate the prevalence of CCPP in some regions of Kenya, Ethiopia, Mauritius, Tajikistan and Pakistan in an international collaborative study.
Results
The strict specificity of the test was confirmed in CCPP-free goat herds exposed to other mycoplasma species of the “mycoides cluster”. Prevalence studies were performed across the enzootic range of the disease in Africa and Asia. Seroprevalence was estimated at 14.6% in the Afar region of Ethiopia, whereas all the herds presented for CCPP vaccination in Kenya tested positive (individual seroprevalence varied from 6 to 90% within each herd). In Mauritius, where CCPP emerged in 2009, nine of 62 herds tested positive. In Central Asia, where the disease was confirmed only recently, no positive animals were detected in the Wakhan District of Afghanistan or across the border in neighboring areas of Tajikistan, whereas seroprevalence varied between 2.7% and 44.2% in the other districts investigated and in northern Pakistan. The test was also used to monitor seroconversion in vaccinated animals.
Conclusions
This newly formatted CCPP cELISA kit has retained the high specificity of the original kit. It can therefore be used to evaluate the prevalence of CCPP in countries or regions without vaccination programs. It could also be used to monitor the efficacy of vaccination campaigns as high-quality vaccines induce high rates of seroconversion.
doi:10.1186/1746-6148-10-48
PMCID: PMC3938968  PMID: 24565080
Contagious caprine pleuropneumonia; Competitive ELISA; Seroprevalence; Kenya; Ethiopia; Mauritius; Tajikistan; Afghanistan; Pakistan; Vaccine quality control
4.  Finishing bacterial genome assemblies with Mix 
BMC Bioinformatics  2013;14(Suppl 15):S16.
Motivation
Among challenges that hamper reaping the benefits of genome assembly are both unfinished assemblies and the ensuing experimental costs. First, numerous software solutions for genome de novo assembly are available, each having its advantages and drawbacks, without clear guidelines as to how to choose among them. Second, these solutions produce draft assemblies that often require a resource intensive finishing phase.
Methods
In this paper we address these two aspects by developing Mix , a tool that mixes two or more draft assemblies, without relying on a reference genome and having the goal to reduce contig fragmentation and thus speed-up genome finishing. The proposed algorithm builds an extension graph where vertices represent extremities of contigs and edges represent existing alignments between these extremities. These alignment edges are used for contig extension. The resulting output assembly corresponds to a set of paths in the extension graph that maximizes the cumulative contig length.
Results
We evaluate the performance of Mix on bacterial NGS data from the GAGE-B study and apply it to newly sequenced Mycoplasma genomes. Resulting final assemblies demonstrate a significant improvement in the overall assembly quality. In particular, Mix is consistent by providing better overall quality results even when the choice is guided solely by standard assembly statistics, as is the case for de novo projects.
Availability
Mix is implemented in Python and is available at https://github.com/cbib/MIX, novel data for our Mycoplasma study is available at http://services.cbib.u-bordeaux2.fr/mix/.
doi:10.1186/1471-2105-14-S15-S16
PMCID: PMC3851838  PMID: 24564706
5.  Characterization of Free Exopolysaccharides Secreted by Mycoplasma mycoides Subsp. mycoides 
PLoS ONE  2013;8(7):e68373.
Contagious bovine pleuropneumonia is a severe respiratory disease of cattle that is caused by a bacterium of the Mycoplasma genus, namely Mycoplasma mycoides subsp. mycoides (Mmm). In the absence of classical virulence determinants, the pathogenicity of Mmm is thought to rely on intrinsic metabolic functions and specific components of the outer cell surface. One of these latter, the capsular polysaccharide galactan has been notably demonstrated to play a role in Mmm persistence and dissemination. The free exopolysaccharides (EPS), also produced by Mmm and shown to circulate in the blood stream of infected cattle, have received little attention so far. Indeed, their characterization has been hindered by the presence of polysaccharide contaminants in the complex mycoplasma culture medium. In this study, we developed a method to produce large quantities of EPS by transfer of mycoplasma cells from their complex broth to a chemically defined medium and subsequent purification. NMR analyses revealed that the purified, free EPS had an identical β(1−>6)-galactofuranosyl structure to that of capsular galactan. We then analyzed intraclonal Mmm variants that produce opaque/translucent colonies on agar. First, we demonstrated that colony opacity was related to the production of a capsule, as observed by electron microscopy. We then compared the EPS extracts and showed that the non-capsulated, translucent colony variants produced higher amounts of free EPS than the capsulated, opaque colony variants. This phenotypic variation was associated with an antigenic variation of a specific glucose phosphotransferase permease. Finally, we conducted in silico analyses of candidate polysaccharide biosynthetic pathways in order to decipher the potential link between glucose phosphotransferase permease activity and attachment/release of galactan. The co-existence of variants producing alternative forms of galactan (capsular versus free extracellular galactan) and associated with an antigenic switch constitutes a finely tuned mechanism that may be involved in virulence.
doi:10.1371/journal.pone.0068373
PMCID: PMC3711806  PMID: 23869216
6.  Draft Genome Sequences of Mycoplasma auris and Mycoplasma yeatsii, Two Species of the Ear Canal of Caprinae 
Genome Announcements  2013;1(3):e00280-13.
We report here the draft genome sequences of Mycoplasma auris and Mycoplasma yeatsii, two species commonly isolated from the external ear canal of Caprinae.
doi:10.1128/genomeA.00280-13
PMCID: PMC3707572  PMID: 23766401
7.  Draft Genome Sequences of Mycoplasma alkalescens, Mycoplasma arginini, and Mycoplasma bovigenitalium, Three Species with Equivocal Pathogenic Status for Cattle 
Genome Announcements  2013;1(3):e00348-13.
We report here the draft genome sequences of Mycoplasma alkalescens, Mycoplasma arginini, and Mycoplasma bovigenitalium. These three species are regularly isolated from bovine clinical specimens, although their role in disease is unclear.
doi:10.1128/genomeA.00348-13
PMCID: PMC3707579  PMID: 23766408
8.  Complete Genome Sequence of Mycoplasma putrefaciens Strain 9231, One of the Agents of Contagious Agalactia in Goats 
Genome Announcements  2013;1(3):e00354-13.
Mycoplasma putrefaciens is one of the etiologic agents of contagious agalactia in goats. We report herein the complete genome sequence of Mycoplasma putrefaciens strain 9231.
doi:10.1128/genomeA.00354-13
PMCID: PMC3707581  PMID: 23766410
9.  Emergence of Atypical Mycoplasma agalactiae Strains Harboring a New Prophage and Associated with an Alpine Wild Ungulate Mortality Episode 
Applied and Environmental Microbiology  2012;78(13):4659-4668.
The bacterium Mycoplasma agalactiae is responsible for contagious agalactia (CA) in small domestic ruminants, a syndrome listed by the World Organization for Animal Health and responsible for severe damage to the dairy industry. Recently, we frequently isolated this pathogen from lung lesions of ibexes during a mortality episode in the French Alps. This situation was unusual in terms of host specificity and tissue tropism, raising the question of M. agalactiae emergence in wildlife. To address this issue, the ibex isolates were characterized using a combination of approaches that included antigenic profiles, molecular typing, optical mapping, and whole-genome sequencing. Genome analyses showed the presence of a new, large prophage containing 35 coding sequences (CDS) that was detected in most but not all ibex strains and has a homolog in Mycoplasma conjunctivae, a species causing keratoconjunctivitis in wild ungulates. This and the presence in all strains of large integrated conjugative elements suggested highly dynamic genomes. Nevertheless, M. agalactiae strains circulating in the ibex population were shown to be highly related, most likely originating from a single parental clone that has also spread to another wild ungulate species of the same geographical area, the chamois. These strains clearly differ from strains described in Europe so far, including those found nearby, before CA eradication a few years ago. While M. agalactiae pathogenicity in ibexes remains unclear, our data showed the emergence of atypical strains in Alpine wild ungulates, raising the question of a role for the wild fauna as a potential reservoir of pathogenic mycoplasmas.
doi:10.1128/AEM.00332-12
PMCID: PMC3370481  PMID: 22522685
10.  Distribution and diversity of mycoplasma plasmids: lessons from cryptic genetic elements 
BMC Microbiology  2012;12:257.
Background
The evolution of mycoplasmas from a common ancestor with Firmicutes has been characterized not only by genome down-sizing but also by horizontal gene transfer between mycoplasma species sharing a common host. The mechanisms of these gene transfers remain unclear because our knowledge of the mycoplasma mobile genetic elements is limited. In particular, only a few plasmids have been described within the Mycoplasma genus.
Results
We have shown that several species of ruminant mycoplasmas carry plasmids that are members of a large family of elements and replicate via a rolling-circle mechanism. All plasmids were isolated from species that either belonged or were closely related to the Mycoplasma mycoides cluster; none was from the Mycoplasma bovis-Mycoplasma agalactiae group. Twenty one plasmids were completely sequenced, named and compared with each other and with the five mycoplasma plasmids previously reported. All plasmids share similar size and genetic organization, and present a mosaic structure. A peculiar case is that of the plasmid pMyBK1 from M. yeatsii; it is larger in size and is predicted to be mobilizable. Its origin of replication and replication protein were identified. In addition, pMyBK1 derivatives were shown to replicate in various species of the M. mycoides cluster, and therefore hold considerable promise for developing gene vectors. The phylogenetic analysis of these plasmids confirms the uniqueness of pMyBK1 and indicates that the other mycoplasma plasmids cluster together, apart from the related replicons found in phytoplasmas and in species of the clade Firmicutes.
Conclusions
Our results unraveled a totally new picture of mycoplasma plasmids. Although they probably play a limited role in the gene exchanges that participate in mycoplasma evolution, they are abundant in some species. Evidence for the occurrence of frequent genetic recombination strongly suggests they are transmitted between species sharing a common host or niche.
doi:10.1186/1471-2180-12-257
PMCID: PMC3541243  PMID: 23145790
Mycoplasma,Plasmid,Replication,Rep protein,Gene transfer,Evolution,Expression vector,Mycoplasma mycoides,Mycoplasma capricolum,Mycoplasma yeatsii
11.  Unexpected genetic diversity of Mycoplasma agalactiae caprine isolates from an endemic geographically restricted area of Spain 
Background
The genetic diversity of Mycoplasma agalactiae (MA) isolates collected in Spain from goats in an area with contagious agalactia (CA) was assessed using a set of validated and new molecular typing methods. Validated methods included pulsed field gel electrophoresis (PFGE), variable number of tandem repeats (VNTR) typing, and Southern blot hybridization using a set of MA DNA probes, including those for typing the vpma genes repertoire. New approaches were based on PCR and targeted genomic regions that diverged between strains as defined by in silico genomic comparisons of sequenced MA genomes.
Results
Overall, the data showed that all typing tools yielded consistent results, with the VNTR analyses being the most rapid method to differentiate the MA isolates with a discriminatory ability comparable to that of PFGE and of a set of new PCR assays. All molecular typing approaches indicated that the Spanish isolates from the endemic area in Murcia were very diverse, with different clonal isolates probably restricted to separate, but geographically close, local areas.
Conclusions
The important genetic diversity of MA observed in infected goats from Spain contrasts with the overall homogeneity of the genomic background encountered in MA from sheep with CA in Southern France or Italy, suggesting that assessment of the disease status in endemic areas may require different approaches in sheep and in goats. A number of congruent sub-typing tools are now available for the differentiation of caprine isolates with comparable discriminatory powers.
doi:10.1186/1746-6148-8-146
PMCID: PMC3514313  PMID: 22920649
Mycoplasma agalactiae; Molecular typing; Contagious agalactia; Goats
12.  A novel substitution matrix fitted to the compositional bias in Mollicutes improves the prediction of homologous relationships 
BMC Bioinformatics  2011;12:457.
Background
Substitution matrices are key parameters for the alignment of two protein sequences, and consequently for most comparative genomics studies. The composition of biological sequences can vary importantly between species and groups of species, and classical matrices such as those in the BLOSUM series fail to accurately estimate alignment scores and statistical significance with sequences sharing marked compositional biases.
Results
We present a general and simple methodology to build matrices that are especially fitted to the compositional bias of proteins. Our approach is inspired from the one used to build the BLOSUM matrices and is based on learning substitution and amino acid frequencies on real sequences with the corresponding compositional bias. We applied it to the large scale comparison of Mollicute AT-rich genomes. The new matrix, MOLLI60, was used to predict pairwise orthology relationships, as well as homolog families among 24 Mollicute genomes. We show that this new matrix enables to better discriminate between true and false orthologs and improves the clustering of homologous proteins, with respect to the use of the classical matrix BLOSUM62.
Conclusions
We show in this paper that well-fitted matrices can improve the predictions of orthologous and homologous relationships among proteins with a similar compositional bias. With the ever-increasing number of sequenced genomes, our approach could prove valuable in numerous comparative studies focusing on atypical genomes.
doi:10.1186/1471-2105-12-457
PMCID: PMC3248887  PMID: 22115330
13.  Mycoplasmoses of ruminants in France: recent data from the national surveillance network 
Background
Ruminant mycoplasmoses are important diseases worldwide and several are listed by the World Organization for Animal Health to be of major economic significance. In France the distribution of mycoplasmal species isolated from clinical samples collected from diseased animals upon veterinary request, is monitored by a network known as VIGIMYC (for VIGIlance to MYCoplasmoses of ruminants). The veterinary diagnostic laboratories collaborating with VIGIMYC are responsible for isolating the mycoplasmas while identification of the isolates is centralized by the French Food Safety Agency (AFSSA) in Lyon. The VIGIMYC framework can also be used for specific surveys and one example, on the prevalence of M. bovis in bovine respiratory diseases, is presented here.
Results
Between 2003 and 2008, 34 laboratories were involved in the network and 1904 mycoplasma isolates, originating from the main ruminant-breeding areas, were identified. For cattle, the high prevalence of M. bovis in bronchopneumonia, notably in young animals, was confirmed by VIGIMYC and an associated specific survey, whereas the non-emergence of species such as M. alkalescens and M. canis was also demonstrated. The etiological agent of bovine contagious pleuropneumonia was never isolated. The principal mycoplasmosis in goats was contagious agalactia with M. mycoides subsp. capri as main agent. Ovine mycoplasmoses, most of which were associated with pneumonia in lambs, were infrequently reported. One exception was ovine contagious agalactia (due to M. agalactiae) that has recently re-emerged in the Pyrénées where it had been endemic for years and was also reported in Corsica, which was previously considered free.
Conclusions
Although VIGIMYC is a passive network and somewhat biased as regards sample collection and processing, it has provided, in this study, an overview of the main mycoplasmoses of ruminants in France. The French epidemiological situation is compared to those existing elsewhere in the world.
doi:10.1186/1746-6148-6-32
PMCID: PMC2892444  PMID: 20525406
14.  Suppression-Subtractive Hybridization as a Strategy To Identify Taxon-Specific Sequences within the Mycoplasma mycoides Cluster: Design and Validation of an M. capricolum subsp. capricolum-Specific PCR Assay▿  
Journal of Clinical Microbiology  2008;46(4):1307-1316.
The phylogenetically related Mycoplasma capricolum subsp. capricolum and M. mycoides subsp. mycoides biotype Large Colony are two small-ruminant pathogens involved in contagious agalactia. Their respective contributions to clinical outbreaks are not well documented, because they are difficult to differentiate with the current diagnostic techniques. In order to identify DNA sequences specific to one taxon or the other, a suppression-subtractive hybridization approach was developed. DNA fragments resulting from the reciprocal subtraction of the type strains were used as probes on a panel of M. capricolum subsp. capricolum and M. mycoides subsp. mycoides biotype Large Colony strains to assess their intrataxon specificity. Due to a high intrataxon polymorphism and important cross-reactions between taxa, a single DNA fragment was shown to be specific for M. capricolum subsp. capricolum and to be present in all M. capricolum subsp. capricolum field isolates tested in this study. A PCR assay targeting the corresponding gene (simpA51) was designed that resulted in a 560-bp amplification only in M. capricolum subsp. capricolum and in M. capricolum subsp. capripneumoniae (the etiological agent of contagious caprine pleuropneumonia). simpA51 was further improved to generate a multiplex PCR (multA51) that allows the differentiation of M. capricolum subsp. capripneumoniae from M. capricolum subsp. capricolum. Both the simpA51 and multA51 assays accurately identify M. capricolum subsp. capricolum among other mycoplasmas, including all members of the M. mycoides cluster. simpA51 and multA51 PCRs are proposed as sensitive and robust tools for the specific identification of M. capricolum subsp. capricolum and M. capricolum subsp. capripneumoniae.
doi:10.1128/JCM.01617-07
PMCID: PMC2292954  PMID: 18234866

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