CrpP Is a Novel Ciprofloxacin-Modifying Enzyme Encoded by the Pseudomonas aeruginosa pUM505 Plasmid.

The pUM505 plasmid, isolated from a clinical isolate, confers resistance to ciprofloxacin (CIP) when transferred into the standard strain PAO1. CIP is an antibiotic of the quinolone family that is used to treat infections. analysis, performed to identify CIP resistance genes, revealed that the 65-amino-acid product encoded by the gene in pUM505 displays 40% amino acid identity to the aminoglycoside phosphotransferase (an enzyme that phosphorylates and inactivates aminoglycoside antibiotics). We cloned (renamed , for iprofloxacin esistance rotein, lasmid encoded) into the pUCP20 shuttle vector. The resulting recombinant plasmid, pUC-, conferred resistance to CIP on strain J53-3, suggesting that this gene encodes a protein involved in CIP resistance. Using coupled enzymatic analysis, we determined that the activity of CrpP on CIP is ATP dependent, while little activity against norfloxacin was detected, suggesting that CIP may undergo phosphorylation. Using a recombinant His-tagged CrpP protein and liquid chromatography-tandem mass spectrometry, we also showed that CIP was phosphorylated prior to its degradation. Thus, our findings demonstrate that CrpP, encoded on the pUM505 plasmid, represents a new mechanism of CIP resistance in , which involves phosphorylation of the antibiotic.