Mesoplasma florum: a near-minimal model organism for systems and synthetic biology.

Mesoplasma florum is an emerging model organism for systems and synthetic biology due to its small genome (∼800 kb) and fast growth rate. While M. florum was isolated and first described almost 40 years ago, many important aspects of its biology have long remained uncharacterized due to technological limitations, the absence of dedicated molecular tools, and since this bacterial species has not been associated with any disease. However, the publication of the first M. florum genome in 2004 paved the way for a new era of research fueled by the rise of systems and synthetic biology. Some of the most important studies included the characterization and heterologous use of M. florum regulatory elements, the development of the first replicable plasmids, comparative genomics and transposon mutagenesis, whole-genome cloning in yeast, genome transplantation, in-depth characterization of the M. florum cell, as well as the development of a high-quality genome-scale metabolic model. The acquired data, knowledge, and tools will greatly facilitate future genome engineering efforts in M. florum, which could next be exploited to rationally design and create synthetic cells to advance fundamental knowledge or for specific applications.

A toolbox for manipulating the genome of the major goat pathogen, Mycoplasma capricolum subsp. capripneumoniae.

Mycoplasma capricolum subspecies capripneumoniae (Mccp) is the causative agent of contagious caprine pleuropneumonia (CCPP), a devastating disease listed by the World Organisation for Animal Health (WOAH) as a notifiable disease and threatening goat production in Africa and Asia. Although a few commercial inactivated vaccines are available, they do not comply with WOAH standards and there are serious doubts regarding their efficacy. One of the limiting factors to comprehend the molecular pathogenesis of CCPP and develop improved vaccines has been the lack of tools for Mccp genome engineering. In this work, key synthetic biology techniques recently developed for closely related mycoplasmas were adapted to Mccp. CReasPy-Cloning was used to simultaneously clone and engineer the Mccp genome in yeast, prior to whole-genome transplantation into M. capricolum subsp. capricolum recipient cells. This approach was used to knock out an S41 serine protease gene recently identified as a potential virulence factor, leading to the generation of the first site-specific Mccp mutants. The Cre-lox recombination system was then applied to remove all DNA sequences added during genome engineering. Finally, the resulting unmarked S41 serine protease mutants were validated by whole-genome sequencing and their non-caseinolytic phenotype was confirmed by casein digestion assay on milk agar. The synthetic biology tools that have been successfully implemented in Mccp allow the addition and removal of genes and other genetic features for the construction of seamless targeted mutants at ease, which will pave the way for both the identification of key pathogenicity determinants of Mccp and the rational design of novel, improved vaccines for the control of CCPP.

Comparative genomics of Mycoplasma feriruminatoris, a fast-growing pathogen of wild Caprinae.

Mycoplasma feriruminatoris is a fast-growing Mycoplasma species isolated from wild Caprinae and first described in 2013. M. feriruminatoris isolates have been associated with arthritis, kerato conjunctivitis, pneumonia and septicemia, but were also recovered from apparently healthy animals. To better understand what defines this species, we performed a genomic survey on 14 strains collected from free-ranging or zoo-housed animals between 1987 and 2017, mostly in Europe. The average chromosome size of the M. feriruminatoris strains was 1,040±0,024 kbp, with 24 % G+C and 852±31 CDS. The core genome and pan-genome of the M. feriruminatoris species contained 628 and 1312 protein families, respectively. The M. feriruminatoris strains displayed a relatively closed pan-genome, with many features and putative virulence factors shared with species from the M. mycoides cluster, including the MIB-MIP Ig cleavage system, a repertoire of DUF285 surface proteins and a complete biosynthetic pathway for galactan. M. feriruminatoris genomes were found to be mostly syntenic, although repertoires of mobile genetic elements, including Mycoplasma Integrative and Conjugative Elements, insertion sequences, and a single plasmid varied. Phylogenetic- and gene content analyses confirmed that M. feriruminatoris was closer to the M. mycoides cluster than to the ruminant species M. yeatsii and M. putrefaciens. Ancestral genome reconstruction showed that the emergence of the M. feriruminatoris species was associated with the gain of 17 gene families, some of which encode defence enzymes and surface proteins, and the loss of 25 others, some of which are involved in sugar transport and metabolism. This comparative study suggests that the M. mycoides cluster could be extended to include M. feriruminatoris. We also find evidence that the specific organization and structure of the DnaA boxes around the oriC of M. feriruminatoris may contribute to drive the remarkable fast growth of this minimal bacterium.

Genome Engineering of the Fast-Growing Mycoplasma feriruminatoris toward a Live Vaccine Chassis.

Development of a new generation of vaccines is a key challenge for the control of infectious diseases affecting both humans and animals. Synthetic biology methods offer new ways to engineer bacterial chassis that can be used as vectors to present heterologous antigens and train the immune system against pathogens. Here, we describe the construction of a bacterial chassis based on the fast-growing Mycoplasma feriruminatoris, and the first steps toward its application as a live vaccine against contagious caprine pleuropneumonia (CCPP). To do so, the M. feriruminatoris genome was cloned in yeast, modified by iterative cycles of Cas9-mediated deletion of loci encoding virulence factors, and transplanted back in Mycoplasma capricolum subsp. capricolum recipient cells to produce the designed M. feriruminatoris chassis. Deleted genes encoded the glycerol transport and metabolism systems GtsABCD and GlpOKF and the Mycoplasma Ig binding protein-Mycoplasma Ig protease (MIB-MIP) immunoglobulin cleavage system. Phenotypic assays of the M. feriruminatoris chassis confirmed the corresponding loss of H2O2 production and IgG cleavage activities, while growth remained unaltered. The resulting mycoplasma chassis was further evaluated as a platform for the expression of heterologous surface proteins. A genome locus encoding an inactivated MIB-MIP system from the CCPP-causative agent Mycoplasma capricolum subsp. capripneumoniae was grafted in replacement of its homolog at the original locus in the chassis genome. Both heterologous proteins were detected in the resulting strain using proteomics, confirming their expression. This study demonstrates that advanced genome engineering methods are henceforth available for the fast-growing M. feriruminatoris, facilitating the development of novel vaccines, in particular against major mycoplasma diseases.

An unequivocal superbug: PDR Klebsiella pneumoniae with an arsenal of resistance and virulence factor genes.

INTRODUCTION: Infections caused by extensively-drug resistant (XDR) and pan-drug resistant (PDR) Klebsiella pneumoniae represent an emerging threat due to the high associated mortality. This study aimed to characterize two carbapenem resistant K. pneumoniae strains from the same patient, the first being PDR (referred to as IMP 1078b) and the second being XDR (referred to IMP 1078s) isolated from the same patient. METHODOLOGY: Antimicrobial susceptibility testing was done for the 2 K. pneumoniae isolates, followed by carbapenem/ß-lactamase inhibitor combination assay, and fitness cost against cefepime and meropenem. Then, whole-genome sequence analysis was performed to decipher the molecular mechanisms behind the high level of resistance recorded in both isolates. Finally, qRT-PCR was done for ß-lactam resistant genes. RESULTS: This is the first report about a K. pneumoniae isolate harboring 47 antimicrobial resistance genes and having type IV pilli (Yersinia) and the fimbrial adherence determinant Stb (Salmonella) as virulence factors. Further analysis on both isolates are discussed within the article. CONCLUSION: The co-existence of a high number of antimicrobial resistant (AMR) genes and virulence factor genes may lead to a life threatening invasive and untreatable infection.

[Synthetic chromosomes: rewriting the code of life]. / Chromosomes synthétiques - Réécrire le code de la vie.

The past decade has seen vast improvements in DNA synthesis and assembly methods. The creation of synthetic DNA molecules is becoming easier and more affordable, such that entire chromosomes can now be synthesized. These advances mark the beginning of synthetic genomics, a new discipline interested in the construction of complete genomes tailored for the study and application of biological systems. From viral genome synthesis to the reconstruction of the yeast 16 chromosomes, we discuss the main discoveries, the regulations and ethical considerations along with the potential of this emerging discipline for the future.

TITLE: Chromosomes synthétiques - Réécrire le code de la vie. ABSTRACT: 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.

[Whole genome transplantation: bringing natural or synthetic bacterial genomes back to life]. / La transplantation de génomes - Redonner vie à des génomes bactériens naturels ou synthétiques.

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.

TITLE: La transplantation de génomes - Redonner vie à des génomes bactériens naturels ou synthétiques. ABSTRACT: 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.

Inferring the Minimal Genome of Mesoplasma florum by Comparative Genomics and Transposon Mutagenesis.

The creation and comparison of minimal genomes will help better define the most fundamental mechanisms supporting life. Mesoplasma florum is a near-minimal, fast-growing, nonpathogenic bacterium potentially amenable to genome reduction efforts. In a comparative genomic study of 13 M. florum strains, including 11 newly sequenced genomes, we have identified the core genome and open pangenome of this species. Our results show that all of the strains have approximately 80% of their gene content in common. Of the remaining 20%, 17% of the genes were found in multiple strains and 3% were unique to any given strain. On the basis of random transposon mutagenesis, we also estimated that ~290 out of 720 genes are essential for M. florum L1 in rich medium. We next evaluated different genome reduction scenarios for M. florum L1 by using gene conservation and essentiality data, as well as comparisons with the first working approximation of a minimal organism, Mycoplasma mycoides JCVI-syn3.0. Our results suggest that 409 of the 473 M. mycoides JCVI-syn3.0 genes have orthologs in M. florum L1. Conversely, 57 putatively essential M. florum L1 genes have no homolog in M. mycoides JCVI-syn3.0. This suggests differences in minimal genome compositions, even for these evolutionarily closely related bacteria. IMPORTANCE The last years have witnessed the development of whole-genome cloning and transplantation methods and the complete synthesis of entire chromosomes. Recently, the first minimal cell, Mycoplasma mycoides JCVI-syn3.0, was created. Despite these milestone achievements, several questions remain to be answered. For example, is the composition of minimal genomes virtually identical in phylogenetically related species? On the basis of comparative genomics and transposon mutagenesis, we investigated this question by using an alternative model, Mesoplasma florum, that is also amenable to genome reduction efforts. Our results suggest that the creation of additional minimal genomes could help reveal different gene compositions and strategies that can support life, even within closely related species.

Cloning and Transplantation of the Mesoplasma florum Genome.

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 the use of genome donor and recipient cell belonging to different species. 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.

Development of oriC-Based Plasmids for Mesoplasma florum.

The near-minimal bacterium Mesoplasma florum constitutes an attractive model for systems biology and for the development of a simplified cell chassis in synthetic biology. However, the lack of genetic engineering tools for this microorganism has limited our capacity to understand its basic biology and modify its genome. To address this issue, we have evaluated the susceptibility of M. florum to common antibiotics and developed the first generation of artificial plasmids able to replicate in this bacterium. Selected regions of the predicted M. florum chromosomal origin of replication (oriC) were used to create different plasmid versions that were tested for their transformation frequency and stability. Using polyethylene glycol-mediated transformation, we observed that plasmids harboring both rpmH-dnaA and dnaA-dnaN intergenic regions, interspaced or not with a copy of the dnaA gene, resulted in a frequency of ∼4.1 × 10-6 transformants per viable cell and were stably maintained throughout multiple generations. In contrast, plasmids containing only one M. florumoriC intergenic region or the heterologous oriC region of Mycoplasma capricolum, Mycoplasma mycoides, or Spiroplasma citri failed to produce any detectable transformants. We also developed alternative transformation procedures based on electroporation and conjugation from Escherichia coli, reaching frequencies up to 7.87 × 10-6 and 8.44 × 10-7 transformants per viable cell, respectively. Finally, we demonstrated the functionality of antibiotic resistance genes active against tetracycline, puromycin, and spectinomycin/streptomycin in M. florum Taken together, these valuable genetic tools will facilitate efforts toward building an M. florum-based near-minimal cellular chassis for synthetic biology.IMPORTANCEMesoplasma florum constitutes an attractive model for systems biology and for the development of a simplified cell chassis in synthetic biology. M. florum is closely related to the mycoides cluster of mycoplasmas, which has become a model for whole-genome cloning, genome transplantation, and genome minimization. However, M. florum shows higher growth rates than other Mollicutes, has no known pathogenic potential, and possesses a significantly smaller genome that positions this species among some of the simplest free-living organisms. So far, the lack of genetic engineering tools has limited our capacity to understand the basic biology of M. florum in order to modify its genome. To address this issue, we have evaluated the susceptibility of M. florum to common antibiotics and developed the first artificial plasmids and transformation methods for this bacterium. This represents a strong basis for ongoing genome engineering efforts using this near-minimal microorganism.

Impact of donor-recipient phylogenetic distance on bacterial genome transplantation.

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.

A Small-Volume, Low-Cost, and Versatile Continuous Culture Device.

BACKGROUND: Continuous culture devices can be used for various purposes such as establishing reproducible growth conditions or maintaining cell populations under a constant environment for long periods. However, commercially available instruments are expensive, were not designed to handle small volumes in the milliliter range, and can lack the flexibility required for the diverse experimental needs found in several laboratories. METHODOLOGY/PRINCIPAL FINDINGS: We developed a versatile continuous culture system and provide detailed instructions as well as a graphical user interface software for potential users to assemble and operate their own instrument. Three culture chambers can be controlled simultaneously with the proposed configuration, and all components are readily available from various sources. We demonstrate that our continuous culture device can be used under different modes, and can easily be programmed to behave either as a turbidostat or chemostat. Addition of fresh medium to the culture vessel can be controlled by a real-time feedback loop or simply calibrated to deliver a defined volume. Furthermore, the selected light-emitting diode and photodetector enable the use of phenol red as a pH indicator, which can be used to indirectly monitor the bulk metabolic activity of a cell population rather than the turbidity. CONCLUSIONS/SIGNIFICANCE: This affordable and customizable system will constitute a useful tool in many areas of biology such as microbial ecology as well as systems and synthetic biology.

Complete Genome Sequence of Escherichia coli BW25113.

Escherichia coli BW25113 is the parent strain of the Keio collection comprising nearly 4,000 single-gene deletion mutants. We report the complete 4,631,469-bp genome sequence of this strain and the key variations from the type strain E. coli MG1655.

Complete Genome Sequence of the Mesoplasma florum W37 Strain.

Mesoplasma florum is a small-genome fast-growing mollicute that is an attractive model for systems and synthetic genomics studies. We report the complete 825,824-bp genome sequence of a second representative of this species, M. florum strain W37, which contains 733 predicted open reading frames and 35 stable RNAs.