A genetic manipulation tool based on the GP35 recombinase for targeted gene editing in mycoplasmas of ruminants

Pathogenic ruminant mycoplasmas are major etiological agents in cattle and small ruminants and are responsible for substantial economic losses in the livestock industry. Progress in pathogenesis research and vaccine development has been hampered by a lack of effective genetic tools. The applicability of common genome editing platforms, such as CRISPR, is inherently restricted in these organisms owing to their minimal genomes, the absence of a cell wall, and low homologous recombination efficiency. Although transposon-mediated random mutagenesis and single-base editing are currently used in the editing of bovine mycoplasma, the stochastic nature of transposons, the risk of single-base random deamination, and limitations in editing window selection hinder the genetic manipulation of bovine mycoplasma. Here, we introduce a plasmid-based methodology that employs the GP35 recombinase from bacteriophage SPP1 to mediate long single-stranded DNA (ssDNA) recombineering, thereby enabling precise gene insertions and deletions in Mycoplasma bovis, with a positive-editing rate of 77.78% - 100%. This targeted system eliminates the risk of random deamination. Leveraging this tool, we generated a panel of M. bovis mutants affecting metabolic and virulence genes and obtained key insights into Mb0564, identified as a novel adhesin. The 192 to 287 aa region of GP35 is critical for interaction with SSB. Structural conservation analysis further suggested that this GP35-ssDNA editing system possesses a high potential for translation to other ruminant pathogens. Collectively, our approach expands the existing genetic toolkit for M. bovis, advances synthetic biology and M. bovis pathobiology, facilitates vaccine development, and strengthens the control of high-impact livestock diseases in line with the One Health framework.