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Mycoplasmas are minimal bacteria that infect humans, wildlife, and most economically relevant livestock species. Mycoplasma infections cause a large range of chronic inflammatory diseases, eventually leading to death in some animals. Due to the lack of efficient recombination and genome engineering tools for most species, the production of mutant strains for the identification of virulence factors and the development of improved vaccine strains is limited. Here, we demonstrate the adaptation of an efficient Cas9-Base Editor system to introduce targeted mutations into three major pathogenic species that span the phylogenetic diversity of these bacteria: the avian pathogen Mycoplasma gallisepticum and the two most important bovine mycoplasmas, Mycoplasma bovis and Mycoplasma mycoides subsp.

In bacteria, cell shape is determined and maintained through a complex interplay between the peptidoglycan cell wall and cytoplasmic filaments made of polymerized MreB. Spiroplasma species, members of the Mollicutes class, challenge this general understanding because they are characterized by a helical cell shape and motility without a cell wall. This specificity is thought to rely on five MreB isoforms and a specific fibril protein. In this study, combinations of these five MreBs and of the fibril from Spiroplasma citri were expressed in another Mollicutes, Mycoplasma capricolum. Mycoplasma cells that were initially pleomorphic, mostly spherical, turned into helices when MreBs and fibrils were expressed in this heterologous host. The fibril protein was essential neither for helicity nor for cell movements. The isoform MreB5 had a special role as it was sufficient to confer helicity and motility to the mycoplasma cells. Cryo-electron microscopy confirmed the association of MreBs and fibril-based cytoskeleton with the plasma membrane, suggesting a direct effect on the membrane curvature. Finally, the heterologous expression of these proteins, MreBs and fibril, made it possible to reproduce the kink-like motility of spiroplasmas without providing the ability of cell movement in liquid broth. We suggest that other Spiroplasma components, not yet identified, are required for swimming, a hypothesis that could be evaluated in future studies using the same model.

Over the past decade, a new strategy was developed to bypass the difficulties to genetically engineer some microbial species by transferring (or "cloning") their genome into another organism that is amenable to efficient genetic modifications and therefore acts as a living workbench. As such, the yeast Saccharomyces cerevisiae has been used to clone and engineer genomes from viruses, bacteria, and algae. The cloning step requires the insertion of yeast genetic elements in the genome of interest, in order to drive its replication and maintenance as an artificial chromosome in the host cell. Current methods used to introduce these genetic elements are still unsatisfactory, due either to their random nature (transposon) or the requirement for unique restriction sites at specific positions (TAR cloning). Here we describe the CReasPy-cloning, a new method that combines both the ability of Cas9 to cleave DNA at a user-specified locus and the yeast's highly efficient homologous recombination to simultaneously clone and engineer a bacterial chromosome in yeast. Using the 0.816 Mbp genome of Mycoplasma pneumoniae as a proof of concept, we demonstrate that our method can be used to introduce the yeast genetic element at any location in the bacterial chromosome while simultaneously deleting various genes or group of genes. We also show that CReasPy-cloning can be used to edit up to three independent genomic loci at the same time with an efficiency high enough to warrant the screening of a small (

MIB and MIP bind to antibodies and disrupt the antigen binding site to promote the dissociation of antibody-antigen interactions., Mycoplasma immunoglobulin binding (MIB) and mycoplasma immunoglobulin protease (MIP) are surface proteins found in the majority of mycoplasma species, acting sequentially to capture antibodies and cleave off their VH domains. Cryo–electron microscopy structures show how MIB and MIP bind to a Fab fragment in a “hug of death” mechanism. As a result, the orientation of the VL and VH domains is twisted out of alignment, disrupting the antigen binding site. We also show that MIB-MIP has the ability to promote the dissociation of the antibody-antigen complex. This system is functional in cells and protects mycoplasmas from antibody-mediated agglutination. These results highlight the key role of the MIB-MIP system in immunity evasion by mycoplasmas through an unprecedented mechanism, and open exciting perspectives to use these proteins as potential tools in the antibody field.

Yeast cells have long been used as hosts to propagate exogenous DNA. Recent progress in genome editing opens new avenues in synthetic biology. These developments allow the efficient engineering of microbial genomes in Saccharomyces cerevisiae that can then be rescued to yield modified bacteria/viruses. Recent examples show that the ability to quickly synthesize, assemble and/or modify viral and bacterial genomes may be a critical factor to respond to emerging pathogens. However, this process has some limitations. DNA molecules much larger than two megabase pairs are complex to clone, bacterial genomes have proven difficult to rescue, and the dual-use potential of these technologies must be carefully considered. Regardless, the use of yeast as a factory has enormous appeal for biological applications., Graphical abstract Image 1, Highlights • Saccharomyces cerevisiae is a robust and efficient host for cloning microbial genomes. • Recent developments allow the efficient engineering of microbial genomes in yeast that can then be rescued using transplantation/transfection to yield modified bacteria/viruses. • Overcoming bottlenecks is key to expand use of yeast as a factory and to offer new possibilities in the synthetic biology field. • Rapid synthesis, assembly or modification of viral and bacterial genomes may be a critical factor to respond to emerging pathogens.

Genome engineering of microorganisms has become a standard in microbial biotechnologies. Several efficient tools are available for the genetic manipulation of model bacteria such as Escherichia coli and Bacillus subtilis, or the yeast Saccharomyces cerevisiae. Difficulties arise when transferring these tools to nonmodel organisms. Synthetic biology strategies relying on genome transplantation (GT) aim at using yeast cells for engineering bacterial genomes cloned as artificial chromosomes. However, these strategies remain unsuccessful for many bacteria, including Mycoplasma pneumoniae (MPN), a human pathogen infecting the respiratory tract that has been extensively studied as a model for systems biology of simple unicellular organisms. Here, we have designed a novel strategy for genome engineering based on the recombinase-assisted genomic engineering (RAGE) technology for editing the MPN genome. Using this strategy, we have introduced a 15 kbp fragment at a specific locus of the MPN genome and replaced 38 kbp from its genome by engineered versions modified either in yeast or in E. coli. A strain harboring a synthetic version of this fragment cleared of 13 nonessential genes could also be built and propagated in vitro. These strains were depleted of known virulence factors aiming at creating an avirulent chassis for SynBio applications. Such a chassis and technology are a step forward to build vaccines or deliver therapeutic compounds in the lungs to prevent or cure respiratory diseases in humans. The authors thank Clara Blanchard for English proofreading. We also thank Dr. Don L. Court for providing biological material and advice. We acknowledge the financial support by the European Union’s Horizon 2020 research and innovation program under Grant Agreement [No. 634942]; and the European MiniCell project selected by ANR, in the frame of the ERASynBio second Joint Call for Transnational Research Projects [No. ANR-15-SYNB-0001-04].

UMR BFP - Equipe Mollicutes; International audience; Cloning and transplantation of bacterial genomes is a powerful method for the creation of engineered microorganisms. However, much remains to be understood about the molecular mechanisms and limitations of this approach. We report the whole-genome cloning of Mesoplasma florum in Saccharomyces cerevisiae, and use this model to investigate the impact of a bacterial chromosome in yeast cells. Our results indicate that the cloned M. florum genome is subjected to weak transcriptional activity, and causes no significant impact on yeast growth. We also report that the M. florum genome can be transplanted into Mycoplasma capricolum without any negative impact from the putative restriction enzyme encoding gene mfl307. Using whole-genome sequencing, we observed that a small number of mutations appeared in all M. florum transplants. Mutations also arose, albeit at a lower frequency, when the M. capricolum genome was transplanted into M. capricolum recipient cells. These observations suggest that genome transplantation is mutagenic, and that this phenomenon is magnified by increased phylogenetic distance between the genome donor and the recipient cell. No difference in efficiency was detected after three successive rounds of genome transplantation, suggesting that the observed mutations were not selected during the procedure. Taken together, our results provide a more accurate picture of the events taking place during bacterial genome cloning and transplantation.

The development of synthetic genomics (SG) allowed the emergence of several groundbreaking techniques including the synthesis, assembly and engineering of whole bacterial genomes. The successful implantation of those methods, which culminated in the creation of JCVI-syn3.0 the first nearly minimal bacterium with a synthetic genome, mainly results from the use of the yeast Saccharomyces cerevisiae as a transient host for bacterial genome replication and modification. Another method played a key role in the resounding success of this project: bacterial genome transplantation (GT). GT consists in the transfer of bacterial genomes cloned in yeast, back into a cellular environment suitable for the expression of their genetic content. While successful using many mycoplasma species, a complete understanding of the factors governing GT will most certainly help unleash the power of the entire SG pipeline to other genetically intractable bacteria.La transplantation de génomes - Redonner vie à des génomes bactériens naturels ou synthétiques.Le développement de la génomique synthétique (GS) a permis l’élaboration d’outils et de méthodes innovantes permettant la synthèse, l’assemblage et la modification génétique précise de chromosomes bactériens complets. La raison principale de ce succès, ayant abouti à la création de la première cellule synthétique quasi-minimale JCVI-syn3.0, est l’utilisation de la levure Saccharomyces cerevisiae comme hôte temporaire d’accueil et de modification de ces génomes. Cependant, une autre technique a joué un rôle considérable dans le succès retentissant de ces travaux : la transplantation de génomes bactériens (TG). Cette technique, encore mal comprise, permet d’installer des génomes complets naturels ou synthétiques dans un contexte cellulaire favorable à leur expression et donner la vie. Une meilleure compréhension du processus de TG permettrait d’élargir l’ensemble des techniques de GS, appliquées actuellement quasi exclusivement à l’étude des mycoplasmes, à de nombreuses autres bactéries d’intérêt, y compris des bactéries génétiquement non-modifiables à ce jour.

Depuis les dix dernières années, les techniques de synthèse et d’assemblage d’ADN se sont grandement améliorées. La construction de molécules d’ADN synthétiques devient maintenant beaucoup plus simple et abordable de sorte qu’il est possible de reconstruire des chromosomes synthétiques complets. Nous assistons donc aux débuts de la génomique synthétique, qui vise la construction de génomes conçus sur mesure pour l’étude et l’utilisation de systèmes biologiques. De la synthèse des premiers génomes viraux jusqu’à la reconstruction des seize chromosomes de la levure, en passant par la première cellule bactérienne contrôlée par un génome entièrement synthétique, nous discutons des découvertes majeures, des aspects réglementaires et éthiques ainsi que du potentiel de cette nouvelle discipline pour le futur.

Mycoplasma hominis is an opportunistic human pathogen associated with genital and neonatal infections. Until this study, the lack of a reliable transformation method for the genetic manipulation of M. hominis hindered the investigation of the pathogenicity and the peculiar arginine-based metabolism of this bacterium. A genomic analysis of 20 different M. hominis strains revealed a number of putative restriction-modification systems in this species. Despite the presence of these systems, a reproducible polyethylene glycol (PEG)-mediated transformation protocol was successfully developed in this study for three different strains: two clinical isolates and the M132 reference strain. Transformants were generated by transposon mutagenesis with an efficiency of approximately 10−9 transformants/cell/µg plasmid and were shown to carry single or multiple mini-transposons randomly inserted within their genomes. One M132-mutant was observed to carry a single-copy transposon inserted within the gene encoding P75, a protein potentially involved in adhesion. However, no difference in adhesion was observed in cell-assays between this mutant and the M132 parent strain. Whole genome sequencing of mutants carrying multiple copies of the transposon further revealed the occurrence of genomic rearrangements. Overall, this is the first time that genetically modified strains of M. hominis have been obtained by random mutagenesis using a mini-transposon conferring resistance to tetracycline.

Many Mycoplasma species have been shown to possess capsular polysaccharides (CPSs). We challenged goats with a CPS-defective or its CPS-possessing parental Mycoplasma mycoides strain. The CPS-defective strain was attenuated, demonstrating that the CPS consisting of galactofuranose is a virulence trait., Capsular polysaccharides have been confirmed to be an important virulence trait in many gram-positive and gram-negative bacteria. Similarly, they are proposed to be virulence traits in minimal Mycoplasma that cause disease in humans and animals. In the current study, goats were infected with the caprine pathogen Mycoplasma mycoides subsp. capri or an engineered mutant lacking the capsular polysaccharide, galactofuranose. Goats infected with the mutant strain showed only transient fever. In contrast, 5 of 8 goats infected with the parental strain reached end-point criteria after infection. These findings confirm that galactofuranose is a virulence factor in M. mycoides.

Mycoplasmas are the smallest free-living organisms and cause a number of economically important diseases affecting humans, animals, insects and plants. Here, we demonstrate that highly virulentMycoplasma mycoidessubspeciescapri(Mmc) can be fully attenuatedviatargeted deletion of non-essential genes encoding, among others, potential virulence traits. Five genomic regions, representing approximately ten percent of the originalMmcgenome, were successively deleted usingSaccharomyces cerevisiaeas an engineering platform. Specifically, a total of 68 genes out of the 432 genes verified to be individually nonessential in the JCVI-Syn3.0 minimal cell, were excised from the genome.In vitrocharacterization showed that this mutant was similar to its parental strain in terms of its doubling time, even though ten percent of the genome content were removed. A novelin vivochallenge model in goats revealed that the wild-type parental strain caused marked necrotizing inflammation at the site of inoculation, septicemia and all animals reaching endpoint criteria within seven days after experimental infection. This is in contrast to the mutant strain, which caused no clinical signs nor pathomorphological lesions. These results highlight, for the first time, the rational design, construction and complete attenuation of aMycoplasmastrain via synthetic genomics tools. Trait addition using the yeast-based genome engineering platform and subsequentin vitroorin vivotrials employing theMycoplasmachassis will allow us to dissect the role of individual candidateMycoplasmavirulence factors and lead the way for the development of an attenuated designer vaccine.IMPORTANCEMembers of theMycoplasma mycoidescluster cause important animal plaques in Africa and Asia, which impact animal welfare, provision of food and the life of thousands of small-scale farmers. We applied synthetic biology tools toMycoplasma mycoidesin order to design and create a fully attenuatedMycoplasmastrain that was subsequently confirmedin vivousing a novel caprine infection model. This is the first time that aMycoplasmamutant developed by applying synthetic biology tools has been testedin vivoto pin point candidate virulence traits. The mutant strain is similar to “apathogenicE. coliK12” strains that boosted the research on host-pathogen interactions for the genusEscherichiaand other bacterial genera.

Mycoplasma species are responsible for several economically significant livestock diseases for which there is a need for new and improved vaccines. Most of the existing mycoplasma vaccines are attenuated strains that have been empirically obtained by serial passages or by chemical mutagenesis. The recent development of synthetic biology approaches has opened the way for the engineering of live mycoplasma vaccines. Using these tools, the essential GTPase-encoding gene obg was modified directly on the Mycoplasma mycoides subsp. capri genome cloned in yeast, reproducing mutations suspected to induce a temperature-sensitive (TS+) phenotype. After transplantation of modified genomes into a recipient cell, the phenotype of the resulting M. mycoides subsp. capri mutants was characterized. Single-point obg mutations did not result in a strong TS+ phenotype in M. mycoides subsp. capri, but a clone presenting three obg mutations was shown to grow with difficulty at temperatures of ≥40°C. This particular mutant was then tested in a caprine septicemia model of M. mycoides subsp. capri infection. Five out of eight goats infected with the parental strain had to be euthanized, in contrast to one out of eight goats infected with the obg mutant, demonstrating an attenuation of virulence in the mutant. Moreover, the strain isolated from the euthanized animal in the group infected with the obg mutant was shown to carry a reversion in the obg gene associated with the loss of the TS+ phenotype. This study demonstrates the feasibility of building attenuated strains of mycoplasma that could contribute to the design of novel vaccines with improved safety. IMPORTANCE Animal diseases due to mycoplasmas are a major cause of morbidity and mortality associated with economic losses for farmers all over the world. Currently used mycoplasma vaccines exhibit several drawbacks, including low efficacy, short time of protection, adverse reactions, and difficulty in differentiating infected from vaccinated animals. Therefore, there is a need for improved vaccines to control animal mycoplasmoses. Here, we used genome engineering tools derived from synthetic biology approaches to produce targeted mutations in the essential GTPase-encoding obg gene of Mycoplasma mycoides subsp. capri. Some of the resulting mutants exhibited a marked temperature-sensitive phenotype. The virulence of one of the obg mutants was evaluated in a caprine septicemia model and found to be strongly reduced. Although the obg mutant reverted to a virulent phenotype in one infected animal, we believe that these results contribute to a strategy that should help in building new vaccines against animal mycoplasmoses.

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UMR BFP - Equipe Mollicutes; International audience; Bacteria of the genus Mycoplasma have recently attracted considerable interest as model organisms in synthetic and systems biology. In particular, Mycoplasma pneumoniae is one of the most intensively studied organisms in the field of systems biology. However, the genetic manipulation of these bacteria is often difficult due to the lack of efficient genetic systems and some intrinsic peculiarities such as an aberrant genetic code. One major disadvantage in working with M. pneumoniae is the lack of replicating plasmids that can be used for the complementation of mutants and the expression of proteins. In this study, we have analysed the genomic region around the gene encoding the replication initiation protein, DnaA, and detected putative binding sites for DnaA (DnaA boxes) that are, however, less conserved than in other bacteria. The construction of several plasmids encompassing this region allowed the selection of plasmid pGP2756 that is stably inherited and that can be used for genetic experiments, as shown by the complementation assays with the glpQ gene encoding the glycerophosphoryl diester phosphodiesterase. Plasmid-borne complementation of the glpQ mutant restored the formation of hydrogen peroxide when bacteria were cultivated in the presence of glycerol phosphocholine. Interestingly, the replicating plasmid can also be used in the close relative, Mycoplasma genitalium but not in more distantly related members of the genus Mycoplasma. Thus, plasmid pGP2756 is a valuable tool for the genetic analysis of M. pneumoniae and M. genitalium.

UMR BFP - Equipe Mollicute; Mollicutes, including mycoplasmas and spiroplasmas, have been considered as good representatives of the « minimal cell » concept: these wall-less bacteria are small in size and possess a minimal genome and restricted metabolic capacities. However, the recent discovery of the presence of post-translational modifications unknown so far, such as the targeted processing of membrane proteins of mycoplasma pathogens for human and swine, revealed a part of the hidden complexity of these microorganisms. In this study, we show that in the phytopathogen, insect-vectored Spiroplasma citri GII-3 adhesion-related protein (ScARP) adhesins are post-translationally processed through an ATP-dependent targeted cleavage. The cleavage efficiency could be enhanced in vitro when decreasing the extracellular pH or upon the addition of polyclonal antibodies directed against ScARP repeated units, suggesting that modification of the surface charge and/or ScARP conformational changes could initiate the cleavage. The two major sites for primary cleavage are localized within predicted disordered regions and do not fit any previously reported cleavage motif; in addition, the inhibition profile and the metal ion requirements indicate that this post-translational modification involves at least one non-conventional protease. Such a proteolytic process may play a role in S. citri colonization of cells of the host insect. Furthermore, our work indicates that post-translational cleavage of adhesins represents a common feature to mollicutes colonizing distinct hosts and that processing of surface antigens could represent a way to make the most out of a minimal genome.

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BFP1332-Equipe Mollicutes; Mycoplasma hominis is a minimal human pathogen that is responsible for genital and neonatal infections. Despite many attempts, there is no efficient genetic tool to manipulate this bacterium, limiting most investigations of its pathogenicity and its uncommon energy metabolism that relies on arginine. The recent cloning and subsequent engineering of other mycoplasma genomes in yeast opens new possibilities for studies of the genomes of genetically intractable organisms. Here, we report the successful one-step cloning of the M. hominis PG21 genome in yeast using the transformation-associated recombination (TAR) cloning method. At low passages, the M. hominis genome cloned into yeast displayed a conserved size. However, after ~60 generations in selective media, this stability was affected, and large degradation events were detected, raising questions regarding the stability of large heterologous DNA molecules cloned in yeast and the need to minimize host propagation. Taking these results into account, we selected early passage yeast clones and successfully modified the M. hominis PG21 genome using the CRISPR/Cas9 editing tool, available in Saccharomyces cerevisiae. Complete M. hominis PG21 genomes lacking the adhesion-related vaa gene were efficiently obtained.

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Mycoplasma mycoides subsp. mycoides is the etiologic agent of contagious bovine pleuropneumonia. We report here the complete genome sequence of the strain T1/44, which is widely used as a live vaccine in Africa.

Genome transplantation (GT) allows the installation of purified chromosomes into recipient cells, causing the resulting organisms to adopt the genotype and the phenotype conferred by the donor cells. This key process remains a bottleneck in synthetic biology, especially for genome engineering strategies of intractable and economically important microbial species. So far, this process has only been reported using two closely related bacteria, Mycoplasma mycoides subsp. capri (Mmc) and Mycoplasma capricolum subsp. capricolum (Mcap), and the main factors driving the compatibility between a donor genome and a recipient cell are poorly understood. Here, we investigated the impact of the evolutionary distance between donor and recipient species on the efficiency of GT. Using Mcap as the recipient cell, we successfully transplanted the genome of six bacteria belonging to the Spiroplasma phylogenetic group but including species of two distinct genera. Our results demonstrate that GT efficiency is inversely correlated with the phylogenetic distance between donor and recipient bacteria but also suggest that other species-specific barriers to GT exist. This work constitutes an important step toward understanding the cellular factors governing the GT process in order to better define and eventually extend the existing genome compatibility limit.

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International audience; Mycoplasma mycoides subsp. capri (Mmc) and subsp. mycoides (Mmm) are important ruminant pathogens worldwide causing diseases such as pleuropneumonia, mastitis and septicaemia. They express galactofuranose residues on their surface, but their role in pathogenesis has not yet been determined. The M. mycoides genomes contain up to several copies of the glf gene, which encodes an enzyme catalyzing the last step in the synthesis of galactofuranose. We generated a deletion of the glf gene in a strain of Mmc using genome transplantation and tandem repeat endonuclease coupled cleavage (TREC) with yeast as an intermediary host for the genome editing. As expected, the resulting YCp1.1-Δglf strain did not produce the galactofuranose containing glycans as shown by immunoblots and immuno-electronmicroscopy employing a galactofuranose specific monoclonal antibody. The mutant lacking galactofuranose exhibited a decreased growth rate and a significantly enhanced adhesion to small ruminant cells. The mutant was also "leaking" as revealed by a β-galactosidase-based assay employing a membrane impermeable substrate. These findings indicate that galactofuranose-containing polysaccharides conceal adhesins and are important for membrane integrity. Unexpectedly, the mutant strain showed increased serum resistance.

International audience; Technologies to synthetically assemble chromosome sized fragments of DNA as well as to enable making thousands of simultaneous changes to existing genomes are now available. These capacities are collectively termed synthetic genomics. The implications of synthetic genomics extend beyond the limited pathway and gene engineering of the past to include the engineering or whole metabolisms, regulatory networks, and even ecosystems. However, in order for those potentials to be met, certain limitations and barriers must be overcome. These barriers no longer include DNA modification and assembly, but instead are based in the limited organisms that many synthetic genomics methods function in, and the limited software for designing custom genomic sequences.

Let There Be Life The DNA sequence information from thousands of genomes is stored digitally as ones and zeros in computer memory. Now, Gibson et al. (p. 52 , published online 20 May; see the cover; see the Policy Forum by Cho and Relman ) have brought together technologies from the past 15 years to start from digital information on the genome of Mycoplasma mycoides to chemically synthesize the genomic DNA as segments that could then be assembled in yeast and transplanted into the cytoplasm of another organism. A number of methods were also incorporated to facilitate testing and error correction of the synthetic genome segments. The transplanted genome became established in the recipient cell, replacing the recipient genome, which was lost from the cell. The reconstituted cells were able to replicate and form colonies, providing a proof-of-principle for future developments in synthetic biology.

Most microbes have not been cultured, and many of those that are cultivatable are difficult, dangerous or expensive to propagate or are genetically intractable. Routine cloning of large genome fractions or whole genomes from these organisms would significantly enhance their discovery and genetic and functional characterization. Here we report the cloning of whole bacterial genomes in the yeast Saccharomyces cerevisiae as single-DNA molecules. We cloned the genomes of Mycoplasma genitalium (0.6 Mb), M. pneumoniae (0.8 Mb) and M. mycoides subspecies capri (1.1 Mb) as yeast circular centromeric plasmids. These genomes appear to be stably maintained in a host that has efficient, well-established methods for DNA manipulation.

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With the development of several new technologies using synthetic biology, it is possible to engineer genetically intractable organisms including Mycoplasma mycoides subspecies capri (Mmc), by cloning the intact bacterial genome in yeast, using the host yeast's genetic tools to modify the cloned genome, and subsequently transplanting the modified genome into a recipient cell to obtain mutant cells encoded by the modified genome. The recently described tandem repeat coupled with endonuclease cleavage (TREC) method has been successfully used to generate seamless deletions and point mutations in the mycoplasma genome using the yeast DNA repair machinery. But, attempts to knock-in genes in some cases have encountered a high background of transformation due to maintenance of unwanted circularization of the transforming DNA, which contains possible autonomously replicating sequence (ARS) activity. To overcome this issue, we incorporated a split marker system into the TREC method, enabling seamless gene knock-in with high efficiency. The modified method is called TREC-assisted gene knock-in (TREC-IN). Since a gene to be knocked-in is delivered by a truncated non-functional marker, the background caused by an incomplete integration is essentially eliminated., In this paper, we demonstrate applications of the TREC-IN method in gene complementation and genome minimization studies in Mmc. In the first example, the Mmc dnaA gene was seamlessly replaced by an orthologous gene, which shares a high degree of identity at the nucleotide level with the original Mmc gene, with high efficiency and low background. In the minimization example, we replaced an essential gene back into the genome that was present in the middle of a cluster of non-essential genes, while deleting the non-essential gene cluster, again with low backgrounds of transformation and high efficiency., Although we have demonstrated the feasibility of TREC-IN in gene complementation and genome minimization studies in Mmc, the applicability of TREC-IN ranges widely. This method proves to be a valuable genetic tool that can be extended for genomic engineering in other genetically intractable organisms, where it may be implemented in elucidating specific metabolic pathways and in rationale vaccine design.

International audience; Efficient protein synthesis in all organisms requires the post-transcriptional methylation of specific ribosomal ribonucleic acid (rRNA) and transfer RNA (tRNA) nucleotides. The methylation reactions are almost invariably catalyzed by enzymes that use S-adenosylmethionine (AdoMet) as the methyl group donor. One noteworthy exception is seen in some bacteria, where the conserved tRNA methylation at m(5)U54 is added by the enzyme TrmFO using flavin adenine dinucleotide together with N(5),N(10)-methylenetetrahydrofolate as the one-carbon donor. The minimalist bacterium Mycoplasma capricolum possesses two homologs of trmFO, but surprisingly lacks the m(5)U54 tRNA modification. We created single and dual deletions of the trmFO homologs using a novel synthetic biology approach. Subsequent analysis of the M. capricolum RNAs by mass spectrometry shows that the TrmFO homolog encoded by Mcap0476 specifically modifies m(5)U1939 in 23S rRNA, a conserved methylation catalyzed by AdoMet-dependent enzymes in all other characterized bacteria. The Mcap0476 methyltransferase (renamed RlmFO) represents the first folate-dependent flavoprotein seen to modify ribosomal RNA.

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The sequenced genome of Mycoplasma mycoides subsp. capri revealed the presence of a Type III restriction–modification system (MmyCI). The methyltransferase (modification) subunit of MmyCI (M.MmyCI) was shown to recognize the sequence 5′-TGAG-3′ and methylate the adenine. The coding region of the methyltransferase gene contains 12 consecutive AG dinucleotide repeats that result in a translational termination at a TAA codon immediately beyond the repeat region. This strain does not have MmyCI activity. A clone was found with 10 AG repeats such that the gene is in frame, and this strain has MmyCI activity, suggesting that the expression of the MmyCI methyltransferase may be phase variable.

Over the past several years, significant advances have been made in the molecular genetics of the Mollicutes (the simplest cells that can be grown in axenic culture). Nevertheless, a number of basic molecular tools are still required before genetic manipulations become routine. Here we describe the development of a new dominant selectable marker based on the enzyme puromycin- N -acetyltransferase from Streptomyces alboniger . Puromycin is an antibiotic that mimics the 3′-terminal end of aminoacylated tRNAs and attaches to the carboxyl terminus of growing protein chains. This stops protein synthesis. Because puromycin conscripts rRNA recognition elements that are used by all of the various tRNAs in a cell, it is unlikely that spontaneous antibiotic resistance can be acquired via a simple point mutation—an annoying issue with existing mycoplasma markers. Our codon-optimized cassette confers pronounced puromycin resistance on all five of the mycoplasma species we have tested so far. The resistance cassette was also designed to function in Escherichia coli , which simplifies the construction of shuttle vectors and makes it trivial to produce the large quantities of DNA generally necessary for mycoplasma transformation. Due to these and other features, we expect the puromycin marker to be a widely applicable tool for studying these simple cells and pathogens.

Character Transplant When engineering bacteria, it can be advantageous to propagate the genomes in yeast. However, to be truly useful, one must be able to transplant the bacterial chromosome from yeast back into a recipient bacterial cell. But because yeast does not contain restriction-modification systems, such transplantation poses problems not encountered in transplantation from one bacterial cell to another. Bacterial genomes isolated after growth in yeast are likely to be susceptible to the restriction-modification system(s) of the recipient cell, as well as their own. Lartigue et al. (p. 1693 , published online 20 August) describe multiple steps, including in vitro DNA methylation, developed to overcome such barriers. A Mycoplasma mycoides large-colony genome was propagated in yeast as a centromeric plasmid, engineered via yeast genetic systems, and, after specific methylation, transplanted into M. capricolum to produce a bacterial cell with the genotype and phenotype of the altered M. mycoides large-colony genome.

As a step toward propagation of synthetic genomes, we completely replaced the genome of a bacterial cell with one from another species by transplanting a whole genome as naked DNA. Intact genomic DNA from Mycoplasma mycoides large colony (LC), virtually free of protein, was transplanted into Mycoplasma capricolum cells by polyethylene glycol–mediated transformation. Cells selected for tetracycline resistance, carried by the M. mycoides LC chromosome, contain the complete donor genome and are free of detectable recipient genomic sequences. These cells that result from genome transplantation are phenotypically identical to the M. mycoides LC donor strain as judged by several criteria.

Replicative oriC plasmids were recently developed for several mollicutes, including three Mycoplasma species belonging to the mycoides cluster that are responsible for bovine and caprine diseases: Mycoplasma mycoides subsp. mycoides small-colony type, Mycoplasma mycoides subsp. mycoides large-colony type, and Mycoplasma capricolum subsp. capricolum . In this study, oriC plasmids were evaluated in M. capricolum subsp. capricolum as genetic tools for (i) expression of heterologous proteins and (ii) gene inactivation by homologous recombination. The reporter gene lacZ , encoding β-galactosidase, and the gene encoding spiralin, an abundant surface lipoprotein of the related mollicute Spiroplasma citri , were successfully expressed. Functional Escherichia coli β-galactosidase was detected in transformed Mycoplasma capricolum subsp. capricolum cells despite noticeable codon usage differences. The expression of spiralin in M. capricolum subsp. capricolum was assessed by colony and Western blotting. Accessibility of this protein at the cell surface and its partition into the Triton X-114 detergent phase suggest a correct maturation of the spiralin precursor. The expression of a heterologous lipoprotein in a mycoplasma raises potentially interesting applications, e.g., the use of these bacteria as live vaccines. Targeted inactivation of gene lppA encoding lipoprotein A was achieved in M. capricolum subsp. capricolum with plasmids harboring a replication origin derived from S. citri . Our results suggest that the selection of the infrequent events of homologous recombination could be enhanced by the use of oriC plasmids derived from related mollicute species. Mycoplasma gene inactivation opens the way to functional genomics in a group of bacteria for which a large wealth of genome data are already available and steadily growing.

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