Development of an oriC vector for use in Mycoplasma synoviae

Abstract

Mycoplasma synoviae, an important poultry pathogen, belonging to the class Mollicutes, causes airsacculitis, synovitis, decreased egg production and produces significant economic losses. Efforts to determine M. synoviae virulence factors and their role in pathogenicity require suitable tools for genetic manipulation of this pathogen. This study describes, for the first time, the identification and cloning of the origin of replication (oriC) of M. synoviae to develop a replicable oriC vector for this mycoplasma. Shuttle vectors containing different putative oriC regions along with tetracycline resistance gene tetM were constructed to transform M. synoviae. An oriC vector, pMAS-LoriC, harbouring the complete dnaA gene along with upstream and downstream DnaA boxes, successfully transformed M. synoviae at an average transformation frequency of 1.07×10(-8) transformants per colony-forming unit (CFU), and remained freely replicating as well as integrated at the chromosomal oriC. Plasmid copy number for pMAS-LoriC was estimated to be 62±29 (average±SD) per cell. This study also provided evidence of the occurrence of homologous recombination and the functionality of the heterologous tetM determinant in M. synoviae. The transformation technique and the oriC vector developed in this study have the potential to be used in targeted gene disruption, gene complementation and expression studies in this organism.

Keywords: Cloning-vector; Electroporation; Gene transfer; Mollicute; Plasmid.