Nucleotide sequence, expression and transcriptional analysis of the Escherichia coli mphK gene encoding macrolide-phosphotransferase K

Escherichia coli 209K harboring the plasmid pSK66, isolated from a Korean patient, is resistant to high levels of erythromycin (MIC > 2 mg/ml) and other macrolide antibiotics. The 2.9 kb fragment derived from the plasmid pSK66 containing mphK (macrolide-phosphotransferase K gene) has been cloned and sequenced. The DNA sequence of mphK is similar to that of mphA from E. coli Tf481A, suggesting that mphK-like genes have been distributed in E. coli by transposition. Nucleotide sequence of the 2.9 kb DNA contain five open reading frames (ORFs). Among them, the mphK gene is 918 bp long, encoding a protein of 306 amino acids with a calculated molecular mass of 33,966 Da. Northern blot analysis reveals that two distinct transcripts are expressed in the 2.9 kb region. The promoter region and the 5′ end of the mphK mRNA have been determined by primer extension analysis. Identity of the gene product as macrolide-phosphotransferase K has been confirmed by expression and purification of the protein. The N-terminal amino acid sequence of the purified MPH(K) has also been found to be identical to that predicted from the gene sequence.