Genome Engineering in Mycoplasma gallisepticum Using Exogenous Recombination Systems

Abstract

Mycoplasma gallisepticum (Mgal) is a common pathogen of poultry worldwide that has recently spread to North American house finches after a single host shift in 1994. The molecular determinants of Mgal virulence and host specificity are still largely unknown, mostly due to the absence of efficient methods for functional genomics. After evaluating two exogenous recombination systems derived from phages found in the phylogenetically related Spiroplasma phoeniceum and the more distant Bacillus subtilis, the RecET-like system from B. subtilis was successfully used for gene inactivation and targeted replacement in Mgal. In a second step, the Cre-lox recombination system was used for the removal of the antibiotic resistance marker in recombinant mutants. This study therefore describes the first genetic tool for targeted genome engineering of Mgal and demonstrates the efficiency of heterologous recombination systems in minimal bacteria.

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

“…Indeed, all genetic elements flanked by the res sequences in the pMT85_2Res can be removed from the chromosome using dedicated resolvase activity ( 57 ), and the oriC plasmids can be lost after a few passages in nonselective medium ( 58 ). Alternatively, CBE constructs can be enhanced with a CRE-Lox system, which is functional in some mycoplasma species ( 23 , 56 , 59 ).…”

“…capricolum ( 89 ) and in M. hyorhinis ( 90 ), but not in M. hyopneumoniae ( 91 ). Recently, a RecET-like system from B. subtilis was used for targeted gene inactivation or gene replacement in Mycoplasma pneumoniae ( 25 ) and M. gallisepticum ( 23 ). However, for now, all of these systems remain limited in efficacy, and their use in a large spectrum of mycoplasmas still needs to be evaluated.…”

“…Only replicative ( oriC ) plasmids and transposon-based mutagenesis are available for M. bovis , and Mmm ( 20 , 21 ). For M. gallisepticum , in addition to oriC plasmids ( 22 ) and transposon mutagenesis, a first tool for the targeted homologous recombination (HR) of short genomic regions has recently been reported ( 23 ).…”

Unblocking genome editing of major animal mycoplasmas using CRISPR/Cas9 base editor systems

“…Indeed, all genetic elements flanked by the res sequences in the pMT85_2 Res can be removed from the chromosome using dedicated resolvase activity (53) and the oriC plasmids such as the pMYSO1 are generally lost after a few passages in non-selective medium (54). Alternatively, CBE constructs can be enhanced with the CRE-Lox system, which is functional in some mycoplasma species (21, 52, 55).…”

“…Only replicative ( oriC ) plasmids and transposon-based mutagenesis are currently available for Mbov , and Mmm (19, 20). For Mgal , a first tool for targeted homologous recombination of short genomic regions has recently been reported (21).…”