Combining Fusion of Cells with CRISPR-Cas9 Editing for the Cloning of Large DNA Fragments or Complete Bacterial Genomes in Yeast.

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 yeast cloning strategies, a new approach combining two already described methods, Fusion cloning and CReasPy-Cloning, was developed. The CReasPy-Fusion method allows the simultaneous cloning and engineering of megabase-sized genomes in yeast by the 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 the cloning efficacy.

Improved transformation efficiency in Mycoplasma hominis enables disruption of the MIB-MIP system targeting human immunoglobulins.

The pathogenicity of Mycoplasma hominis is poorly understood, mainly due to the absence of efficient genetic tools. A polyethylene glycol-mediated transformation protocol was recently developed for the M. hominis reference strain M132 using the pMT85-Tet plasmid. The transformation efficiency remained low, hampering generation of a large mutant library. In this study, we improved transformation efficiency by designing M. hominis-specific pMT85 derivatives. Using the Gibson Assembly, the Enterococcus-derived tet(M) gene of the pMT85-Tet plasmid was replaced by that of a M. hominis clinical isolate. Next, the Spiroplasma-derived spiralin gene promoter driving tet(M) expression was substituted by one of three putative regulatory regions (RRs): the M. hominis arginine deiminase RR, the M. hominis elongation factor Tu RR, or the 68 bp SynMyco synthetic RR. SynMyco-based construction led to a 100-fold increase in transformation efficiency in M. hominis M132. This construct was also transformed into the M. hominis PG21 reference strain and three other clinical isolates. The transposon insertion locus was determined for 128 M132-transformants. The majority of the impacted coding sequences encoded lipoproteins and proteins involved in DNA repair or in gene transfer. One transposon integration site was in the mycoplasma immunoglobulin protease gene. Phenotypic characterization of the mutant showed complete disruption of the human antibody cleavage ability of the transformant. These results demonstrate that our M. hominis-optimized plasmid can be used to generate large random transposon insertion libraries, enabling future studies of the pathogenicity of M. hominis. IMPORTANCE Mycoplasma hominis is an opportunistic human pathogen, whose physiopathology is poorly understood and for which genetic tools for transposition mutagenesis have been unavailable for years. A PEG-mediated transformation protocol was developed using the pMT85-Tet plasmid, but the transformation efficiency remained low. We designed a modified pMT85-Tet plasmid suitable for M. hominis. The use of a synthetic regulatory region upstream of the antibiotic resistance marker led to a 100-fold increase in the transformation efficiency. The generation and characterization of large transposon mutagenesis mutant libraries will provide insight into M. hominis pathogenesis. We selected a transformant in which the transposon was integrated in the locus encoding the immunoglobulin cleavage system MIB-MIP. Phenotypic characterization showed that the wild-type strain has a functional MIB-MIP system, whereas the mutant strain had lost the ability to cleave human immunoglobulins.

Cytoskeletal components can turn wall-less spherical bacteria into kinking helices.

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.

Genome Editing of Veterinary Relevant Mycoplasmas Using a CRISPR-Cas Base Editor System.

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. mycoides. As a proof of concept, we successfully used an inducible SpdCas9-pmcDA1 cytosine 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 powerful enough to disrupt 54 of 55 insertion sequence transposases in a single experiment. Whole-genome sequencing of the edited strains showed that off-target mutations were limited, suggesting 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 likely the entire class Mollicutes. IMPORTANCE 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. The lack of precise genome editing tools has hampered functional studies of many species, leaving multiple questions about the molecular basis of their pathogenicity unanswered. Here, we demonstrate the adaptation of 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 of these economically important bacteria.

Imaging Minimal Bacteria at the Nanoscale: a Reliable and Versatile Process to Perform Single-Molecule Localization Microscopy in Mycoplasmas.

Mycoplasmas are the smallest free-living organisms. These bacteria are important models for both fundamental and synthetic biology, owing to their highly reduced genomes. They are also relevant in the medical and veterinary fields, as they are pathogenic to both humans and most livestock species. Mycoplasma cells have minute sizes, often in the 300- to 800-nm range. As these dimensions are close to the diffraction limit of visible light, fluorescence imaging in mycoplasmas is often poorly informative. Recently developed superresolution imaging techniques can break this diffraction limit, improving the imaging resolution by an order of magnitude and offering a new nanoscale vision of the organization of these bacteria. These techniques have, however, not been applied to mycoplasmas before. Here, we describe an efficient and reliable protocol to perform single-molecule localization microscopy (SMLM) imaging in mycoplasmas. We provide a polyvalent transposon-based system to express the photoconvertible fluorescent protein mEos3.2, enabling photo-activated localization microscopy (PALM) in most Mycoplasma species. We also describe the application of direct stochastic optical reconstruction microscopy (dSTORM). We showcase the potential of these techniques by studying the subcellular localization of two proteins of interest. Our work highlights the benefits of state-of-the-art microscopy techniques for mycoplasmology and provides an incentive to further the development of SMLM strategies to study these organisms in the future. IMPORTANCE Mycoplasmas are important models in biology, as well as highly problematic pathogens in the medical and veterinary fields. The very small sizes of these bacteria, well below a micron, limits the usefulness of traditional fluorescence imaging methods, as their resolution limit is similar to the dimensions of the cells. Here, to bypass this issue, we established a set of state-of-the-art superresolution microscopy techniques in a wide range of Mycoplasma species. We describe two strategies: PALM, based on the expression of a specific photoconvertible fluorescent protein, and dSTORM, based on fluorophore-coupled antibody labeling. With these methods, we successfully performed single-molecule imaging of proteins of interest at the surface of the cells and in the cytoplasm, at lateral resolutions well below 50 nm. Our work paves the way toward a better understanding of mycoplasma biology through imaging of subcellular structures at the nanometer scale.

A RAGE Based Strategy for the Genome Engineering of the Human Respiratory Pathogen Mycoplasma pneumoniae.

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.

Random transposon insertion in the Mycoplasma hominis minimal genome.

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.

Cloning, Stability, and Modification of Mycoplasma hominis Genome in Yeast.

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.