Complementation of the Mycoplasma synoviae MS-H vaccine strain with wild-type oppF 1 influences its growth characteristics.

The Mycoplasma synoviae (MS) vaccine strain MS-H harbours a frameshift mutation in oppF ₁ (oligopeptide permease transporter) which results in expression of a truncated OppF ₁ . The effect of this mutation on growth and attenuation of the MS-H is unknown. In this study, the impact of the mutation on the vaccine phenotype was investigated in vitro by introducing a wild-type copy of oppF ₁ gene in the MS-H genome. Wild-type oppF ₁ was cloned under the vlhA promoter into an oriC vector carrying a tetracycline resistance gene. MS-H was successfully transformed with the final construct pMS-oppF ₁ -tetM or with a similar vector lacking oppF ₁ coding sequence (pMS-tetM). The MS-H transformed with pMS-oppF ₁ -tetM exhibited smaller colony size than MS-H transformed with pMS-tetM. Monospecific rabbit sera against C-terminus of OppF ₁ detected bands of expected size for full-length OppF ₁ in the 86079/7NS parental strain of MS-H and the MS-H transformed with pMS-oppF ₁ -tetM, but not in MS-H and MS-H transformed with pMS-tetM. Comparison of the growth curve of MS-H transformants harvested from media with/without tetracycline was conducted using vlhA Q-PCR which revealed that MS-H transformed with pMS-tetM had a higher growth rate than MS-H transformed with pMS-oppF ₁ -tetM in the media with/without tetracycline. Lastly, the whole genome sequencing of MS-H transformed with pMS-oppF ₁ -tetM (passage 27) showed that the chromosomal copy of the mutated oppF ₁ had been replaced with a wild-type version of the gene. This study reveals that the truncation of oppF ₁ impacts on growth characteristics of the MS-H and provides insight into the molecular pathogenesis of MS and perhaps broader mycoplasma species. RESEARCH HIGHLIGHTS The full-length OppF ₁ was expressed in Mycoplasma synoviae MS-H vaccine.Truncation of oppF ₁ impacts on growth characteristics of the MS-H.Chromosomal copy of the mutated oppF ₁ in MS-H was replaced with wild-type oppF ₁ .