Development of a replicable oriC plasmid for Mycoplasma gallisepticum and Mycoplasma imitans, and gene disruption through homologous recombination in M. gallisepticum

The genome of Mycoplasma gallisepticum strain R(low) has been sequenced completely, but subsequent genetic studies have been limited by the lack of a replicable vector system. In this study, replicable plasmids were constructed for M. gallisepticum and Mycoplasma imitans using the oriC region upstream from the soj gene. The oriC plasmids of M. gallisepticum (pGTLori) and M. imitans (pMIori) replicated in both species, but Mycoplasma pneumoniae could not support replication of pGTLori. A 180 bp section of the oriC region of M. gallisepticum was found to be the minimal region required for plasmid replication in M. gallisepticum strain S6, the shortest oriC region defined for mycoplasmas. Targeted gene disruption of vlhA1.1 of M. gallisepticum S6 was attempted using these oriC plasmids. Constructs made in pPLoriC7 integrated into the M. gallisepticum genomic oriC region, not into the targeted gene, whereas those made in pMIori disrupted the vlhA1.2 gene, which has 97 % DNA sequence identity with the vlhA1.1 gene. During in vitro passages, antimicrobial selection pressure did not influence the rate of chromosomal integration. These oriC plasmids will thus be useful for genetic studies, including inactivation or expression of selected genes, in M. gallisepticum and M. imitans, and will lead to a better understanding of their molecular biology. They are, to our knowledge, the first replicable plasmids developed for the Pneumoniae phylogenetic group of mycoplasmas.