Macrolide 2'-phosphotransferase [MPH(2')] was purified 90-fold from an erythromycin-resistant strain of Escherichia coli, and its enzymatic properties were investigated. MPH(2') is an inducible intracellular enzyme which showed high levels of activity with 14-member-ring macrolides and extremely low levels with 16-member-ring macrolides. The optimum pH for inactivation of oleandomycin was 8.2, and the optimum temperature of the reaction was 40°C. Enzyme activity was lost by heat treatment at 50°C for 1 min. The isoelectric point and molecular weight of the enzyme were 5.3 and 34,000, respectively. Purine nucleotides, such as GTP, ITP, and ATP, were effective as cofactors in the inactivation of macrolides. Iodine, EDTA, or divalent cations inhibited MPH(2') activity.In general, erythromycin is much better known for its activity against gram-positive bacteria and Mycoplasma pneumoniae than for its activity against gram-negative bacteria. However, in Europe, macrolides, in particular erythromycin, have been recommended for prophylaxis of septicemia in immunocompromised patients (1, 2) and for prevention of traveler's diarrhea (4). Recently, members of the family Enterobacteriaceae which were highly resistant to erythromycin were isolated from patients in a hematologyoncology unit (3). Colonization of the intestinal tract by members of the family Enterobacteriaceae that are highly resistant to erythromycin is usually associated with previous long-term oral therapy with the drug (2-4). It has been reported that high-level resistance to erythromycin in members of the family Enterobacteriaceae results from production of erythromycin esterases and rRNA methylases (1,5,(6)(7)(8)24).We reported previously that the structure of inactivated oleandomycin generated by erythromycin-resistant (MIC, 1,600 ,ug/ml) Escherichia coli Tf481A, which was isolated from a patient in Japan in 1983, was oleandomycin 2'-phosphate (22). This paper describes the purification and characterization of a new macrolide-inactivating enzyme, macrolide 2'-phosphotransferase [MPH(2')].
MATERIALS AND METHODSBacterial strains. A clinical isolate of E. coli Tf481A was used for determination of drug resistance and for preparation of crude extracts. Bacillus subtilis ATCC 6633 (20) and Staphylococcus aureus 209P (13) were used as test strains in microbioassays for determination of the potencies of macrolides and lincosamide antibiotics, respectively. E. coli K-12 W3110 Rif' (14), which is susceptible to macrolides, was used as a reference strain.Media. Nutrient broth (Eiken Chemical Co. Ltd., Tokyo, Japan) was used as a liquid medium, and nutrient agar was used as a solid medium.Antibiotics and reagents. Erythromycin, oleandomycin, and spiramycin were purchased from Sigma Chemical Co., * Corresponding author.St. Louis, Mo.; and leucomycin was purchased from Wako Pure Chemical Industries Ltd., Osaka, Japan. Erythromycin A was a gift from Shionogi & Co. Ltd., Osaka, Japan; josamycin was a gift from Yamanouchi Pharmaceutical Co.Ltd., Tokyo, Japan; midecamycin...
