title: Cloning, Stability, and Modification of Mycoplasma hominis Genome in Yeast.
abstract: 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.
authors.affiliation: Univ. Bordeaux , USC-EA3671 Mycoplasmal and Chlamydial Infections in Humans, F-33000 Bordeaux, France.|INRA , USC-EA3671 Mycoplasmal and Chlamydial Infections in Humans, F-33000 Bordeaux, France.
authors.forename: Fabien
authors.initials: F
authors.lastname: Rideau
authors.affiliation: Univ. Bordeaux , USC-EA3671 Mycoplasmal and Chlamydial Infections in Humans, F-33000 Bordeaux, France.|INRA , USC-EA3671 Mycoplasmal and Chlamydial Infections in Humans, F-33000 Bordeaux, France.
authors.forename: Chloé
authors.identifier: 0000-0003-3235-9264
authors.initials: C
authors.lastname: Le Roy
authors.affiliation: Univ. Bordeaux , USC-EA3671 Mycoplasmal and Chlamydial Infections in Humans, F-33000 Bordeaux, France.|INRA , USC-EA3671 Mycoplasmal and Chlamydial Infections in Humans, F-33000 Bordeaux, France.
authors.forename: Elodie C T
authors.initials: ECT
authors.lastname: Descamps
authors.affiliation: Univ. Bordeaux , USC-EA3671 Mycoplasmal and Chlamydial Infections in Humans, F-33000 Bordeaux, France.|INRA , USC-EA3671 Mycoplasmal and Chlamydial Infections in Humans, F-33000 Bordeaux, France.
authors.forename: Hélène
authors.initials: H
authors.lastname: Renaudin
authors.affiliation: INRA , UMR 1332 de Biologie du Fruit et Pathologie, F-33140 Villenave d'Ornon, France.|Univ. Bordeaux , UMR 1332 de Biologie du Fruit et Pathologie, F-33140 Villenave d'Ornon, France.
authors.forename: Carole
authors.initials: C
authors.lastname: Lartigue
authors.affiliation: Univ. Bordeaux , USC-EA3671 Mycoplasmal and Chlamydial Infections in Humans, F-33000 Bordeaux, France.|INRA , USC-EA3671 Mycoplasmal and Chlamydial Infections in Humans, F-33000 Bordeaux, France.
authors.forename: Cécile
authors.initials: C
authors.lastname: Bébéar
mesh_terms: D003001:Cloning, Molecular; D005822:Genetic Vectors / Q000235:genetics; D016680:Genome, Bacterial / Q000235:genetics*; D019535:Mycoplasma hominis / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics
keywords: CRISPR/Cas9; Mycoplasma hominis; Vaa; genome stability; transformation-associated recombination (TAR) cloning; yeast centromeric plasmid