Identification of a chromosomally encoded kanamycin acetylase in Porphyromonas gingivalis
Abstract:
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.
Insights
Sonic extracts from Porphyromonas gingivalis inactivate kanamycin sulfate through acetylation, requiring acetyl coenzyme A (acetyl-CoA). This gene is conserved across P. gingivalis strains, with potential implications for antibiotic resistance research.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Porphyromonas gingivalis is a key pathogen in periodontitis.
- Antibiotic resistance is a growing global health concern.
- Mechanisms of antibiotic inactivation by oral bacteria are not fully understood.
Purpose of the Study:
- To investigate the ability of Porphyromonas gingivalis sonic extracts to inactivate kanamycin sulfate.
- To elucidate the mechanism of kanamycin inactivation.
- To clone and characterize the gene responsible for kanamycin inactivation.
Main Methods:
- Sonic extracts of P. gingivalis, Prevotella intermedia, and Prevotella nigrescens were prepared.
- Kanamycin inactivation was assessed using a bio-assay with Escherichia coli JM109.
- The gene for kanamycin inactivation was cloned into E. coli using plasmid vector PTZ18R.
- Southern blot analysis was performed to assess gene conservation.
Main Results:
- Sonic extracts of all P. gingivalis strains inactivated kanamycin sulfate.
- Kanamycin inactivation by P. gingivalis required acetyl coenzyme A (acetyl-CoA), indicating acetylation.
- A kanamycin-resistant clone expressing inactivation activity was generated by cloning the P. gingivalis gene.
- The gene for kanamycin inactivation was conserved among all tested P. gingivalis strains.
Conclusions:
- Porphyromonas gingivalis possesses a mechanism for kanamycin inactivation via acetylation.
- The gene encoding this kanamycin-inactivating protein is conserved within P. gingivalis.
- Understanding these inactivation mechanisms is crucial for addressing antibiotic resistance.
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