Construction and analysis of a modified Tn4001 conferring chloramphenicol resistance in Mycoplasma pneumoniae

The Staphylococcus aureus transposon Tn4001 and derivatives thereof have been transformed successfully in several mycoplasma species. In order to expand the versatility of Tn4001 for other genetic manipulations and for use in mycoplasma species resistant to gentamicin (Gm), chloramphenicol acetyltransferase (Cat) from S. aureus was evaluated as a selectable marker. The cat gene was cloned in both orientations into a modified Tn4001 and transformed into Mycoplasma pneumoniae, conferring resistance to Cm and Gm. Replacement of the gene for GmR in Tn4001 with cat likewise conferred CmR when transformed into M. pneumoniae. The minimum inhibitory concentration to Cm in transformants with cat derivatives of Tn4001 was 300-500 microg/ml, and Cat enzyme activity was demonstrated by using a fluorescent substrate.