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

The Staphylococcus aureus transposon Tn4001 and derivatives thereof have be en transformed successfully in several mycoplasma species. In order to expa nd the versatility of Tn4001 for other genetic manipulations and for use in mycoplasma species resistant to gentamicin (Gm), chloramphenicol acetyltra nsferase (Cat) from S. aureus was evaluated as a selectable marker. The cat gene was cloned in both orientations into a modified Tn4001 and transforme d into Mycoplasma pneumoniae, conferring resistance to Cm and Gm. Replaceme nt of the gene for Gm(R) in Tn4001 with cut likewise conferred Cm-R when tr ansformed into M. pneumoniae. The minimum inhibitory concentration to Cm in transformants with cat derivatives of Tn4001 was 300-500 mu g/ml, and Cat enzyme activity was demonstrated by using a fluorescent substrate. (C) 1999 Academic Press.