Identification of a chromosomally encoded kanamycin acetylase in Porphyromonas gingivalis.

Sonic extracts from the test strains of Porphyromonas gingivalis, Prevotella intermedia and Prevotella nigrescens were tested for the ability to inactivate kanamycin sulfate in the presence of ATP or acetyl coenzyme A (acetyl-CoA). Residual kanamycin activity was determined by a bio-assay using Escherichia coli JM109 as the assay organism. Sonic extracts of all test strains of P. gingivalis inactivated kanamycin. All strains tested in this study required the presence of acetyl-CoA for inactivation, indicating that inactivation was by acetylation. The gene of a kanamycin-inactivating protein from P. gingivalis 16-1 was cloned into E. coli utilizing the plasmid vector PTZ18R. The resultant kanamycin-resistant clone, harboring plasmid pPG16 with a P. gingivalis insert, expressed a kanamycin-inactivating activity, which was enhanced by the addition of acetyl-CoA, confirming that the kanamycin was inactivated by acetylation. Southern blot analysis indicates that the gene was conserved among all P. gingivalis strains tested.