Alain Blanchard

Mycoplasma bovis is a minimal bacterium infecting cattle, which causes a wide variety of symptoms and is impacting dairy and beef producers worldwide. Part of the difficulty in research surrounding M. bovis , and other mycoplasmas, is the lack of efficient genome editing tools. As a proof of concept, we previously presented a transposon-based CRISPR-Base Editor system to introduce targeted mutations in M. bovis . In this work, the existing tool has been greatly improved: multi-loci targeting through addition of a second guide RNA; increased number of targetable loci by using an engineered Cas9 with AT-rich PAM specificity, and elimination of the CRISPR-Base Editor from the generated mutants through either transposon excision or use of a curable plasmid. We also propose a dedicated bioinformatic tool to identify target sequences in genes of a given genome. This software was applied to demonstrate the potential of our improved tools in M. bovis and other mycoplasmas of veterinary and human interest that currently lack genome editing methods.

Genome-based analysis allows for large-scale classification of diverse bacteria and has been widely adopted for delineating species. Unfortunately, for higher taxonomic ranks such as genus, establishing a generally accepted approach based on genome analysis is challenging. While core-genome phylogenies depict the evolutionary relationships among species, determining the correspondence between clades and genera may not be straightforward. For genotypic divergence, the percentage of conserved proteins and genome-wide average amino acid identity are commonly used, but often do not provide a clear threshold for classification. In this work, we investigated the utility of global comparisons and data visualization in identifying clusters of species based on their overall gene content and rationalized that such patterns can be integrated with phylogeny and other information such as phenotypes for improving taxonomy. As a proof of concept, we selected 177 representative genome sequences from the Mycoplasmatales–Entomoplasmatales clade within the class Mollicutes for a case study. We found that the clustering patterns corresponded to the current understanding of these organisms, namely the split into three above-genus groups: Hominis, Pneumoniae and Spiroplasma–Entomoplasmataceae–Mycoides. However, at the genus level, several important issues were found. For example, recent taxonomic revisions that split the Hominis group into three genera and Entomoplasmataceae into five genera are problematic, as those newly described or emended genera lack clear differentiations in gene content from one another. Moreover, several cases of misclassification were identified. These findings demonstrated the utility of this approach and its potential application to other bacteria.

A corrigendum of this article has been published full details can be found at https://doi.org/10.1099/ijsem.0.006662 This report documents the International Committee on Systematics of Prokaryotes Subcommittee on the Taxonomy of Mollicutes minutes for the meeting held 7 July 2024, Gran Canaria, Spain, at the 25th International Organization for Mycoplasmology Congress.

Genome-based analysis allows for large-scale classification of diverse bacteria and has been widely adopted for delineating species. Unfortunately, for higher taxonomic ranks such as genus, establishing a generally accepted approach based on genome analysis is challenging. While core-genome phylogenies depict the evolutionary relationships among species, determining the correspondence between clades and genera may not be straightforward. For genotypic divergence, percentage of conserved proteins (POCP) and genome-wide average amino acid identity (AAI) are commonly used, but often do not provide a clear threshold for classification. In this work, we investigated the utility of global comparisons and data visualization in identifying clusters of species based on their overall gene content, and rationalized that such patterns can be integrated with phylogeny and other information such as phenotypes for improving taxonomy. As a proof of concept, we selected 177 representative genome sequences from the Mycoplasmatales–Entomoplasmatales clade within the class Mollicutes for a case study. We found that the clustering patterns corresponded to the current understanding of these organisms, namely the split into three above-genus groups: Hominis, Pneumoniae, and Spiroplasma-Entomoplasmataceae-Mycoides (SEM). However, at the genus level, several important issues were found. For example, recent taxonomic revisions that split the Hominis group into three genera and Entomoplasmataceae into five genera are problematic, as those newly described or emended genera lack clear differentiations in gene content from one another. Moreover, several cases of mis-classification were identified. These findings demonstrated the utility of this approach and the potential application for other bacteria. Impact statement Taxonomy provides a foundation for communication that involves biological entities. As such, robust classification and standardized nomenclature are critical. In recent years, genome analysis has been widely adopted for delineating species, but generally accepted approaches for delineating higher taxonomic ranks such as genus are lacking. In this work, we demonstrated that the comparison of overall gene content among species provides an intuitive method for identifying groups of similar organisms that can correspond to genera. Moreover, several critical issues were identified when we applied the method to evaluate recent taxonomic revisions that affected many pathogens with biomedical or economic importance. These findings serve as a cautionary tale against the over-reliance of core-genome centered approaches in taxonomy. Data Summary The 183 genome assemblies analyzed in this study were obtained from the National Center for Biotechnology Information (NCBI) Genome Database. The accession numbers are provided in Table S1.

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.

The genetic engineering of genome fragments larger than 100 kbp is challenging and requires both specific methods and cloning hosts. The yeast Saccharomyces cerevisiae is considered as a host of choice for cloning and engineering whole or partial genomes from viruses, bacteria, and algae. Several methods are now available to perform these manipulations, each with its own limitations. In order to extend the range of in-yeast cloning strategies, a new approach combining two already described methods, the Fusion cloning and the CReasPy-Cloning, was developed. The CReasPy-Fusion method allows the simultaneous cloning and engineering of megabase-sized genomes in yeast by fusion of bacterial cells with yeast spheroplasts carrying the CRISPR-Cas9 system. With this new approach, we demonstrate the feasibility of cloning and editing whole genomes from several Mycoplasma species belonging to different phylogenetic groups. We also show that CReasPy-Fusion allows the capture of large genome fragments with high efficacy, resulting in the successful cloning of selected loci in yeast. We finally identify bacterial nuclease encoding genes as barriers for CReasPy-Fusion by showing that their removal from the donor genome improves cloning efficacy.

Bacterial cell shape is generally determined through an interplay between the peptidoglycan cell wall and cytoplasmic filaments made of polymerized MreB. Indeed, some bacteria (e.g., Mycoplasma) that lack both a cell wall and mreB genes consist of non-motile cells that are spherical or pleomorphic. However, other members of the same class Mollicutes (e.g., Spiroplasma, also lacking a cell wall) display a helical cell shape and kink-based motility, which is thought to rely on the presence of five MreB isoforms and a specific fibril protein. Here, we show that heterologous expression of Spiroplasma fibril and MreB proteins confers helical shape and kinking ability to Mycoplasma capricolum cells. Isoform MreB5 is sufficient to confer helicity and kink propagation to mycoplasma cells. Cryoelectron microscopy confirms the association of cytoplasmic MreB filaments with the plasma membrane, suggesting a direct effect on membrane curvature. However, in our experiments, the heterologous expression of MreBs and fibril did not result in efficient motility in culture broth, indicating that additional, unknown Spiroplasma components are required for swimming.

Mycoplasmas are minimal pathogenic bacteria that infect a wide range of hosts, including humans, livestock, and wild animals. Major pathogenic species cause acute to chronic infections involving still poorly characterized virulence factors.

Mycoplasmas are minimal bacteria that infect humans, wildlife, and most economically important 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, the production of mutant strains for the identification of virulence factors and the development of improved vaccine strains is still a bottleneck for many mycoplasma species. Here, we demonstrate the efficacy of a CRISPR-derived genetic tool to introduce targeted mutations in 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. mycoides . As a proof of concept, we successfully used an inducible dCas9-cytidine deaminase system to disrupt several major virulence factors in these pathogens. Various induction times and inducer concentrations were evaluated to optimize editing efficiency. The optimized system was sufficiently powerful to disrupt 54 of 55 insertion sequence transposases in a single step. Whole genome sequencing showed that off-target mutations were limited and suggest that most variations detected in the edited genomes are Cas9-independent. This effective, rapid, and easy-to-use genetic tool opens a new avenue for the study of these important animal pathogens and, most likely, the entire class Mollicutes . Significance: Mycoplasmas are minimal wall-less pathogenic bacteria that infect a wide range of hosts, including humans, livestock, and wild animals. Major pathogenic species cause acute to chronic infections involving still poorly characterized virulence factors. The lack of precise genome editing tools has hampered functional studies for many species, leaving multiple questions about the molecular basis of their pathogenicity unanswered. We developed a CRISPR-derived base editor for three major pathogenic species, Mycoplasma gallisepticum , Mycoplasma bovis , and Mycoplasma mycoides subsp . mycoides . Several virulence factors were successfully targeted and we were able to edit up to 54 target sites in a single step. The availability of this efficient and easy-to-use genetic tool will greatly facilitate functional studies in these economically important bacteria.