INACTIVATION OF CHLORAMPHENICOL BY O-PHOSPHORYLATION - A NOVEL RESISTANCE MECHANISM IN STREPTOMYCES-VENEZUELAE ISP5230, A CHLORAMPHENICOL PRODUCER

Plasmid pJV4, containing a 2.4-kilobase pair insert of genomic DNA fro m the chloramphenicol (Cm) producer Streptomyces venezuelae ISP5230, c onfers resistance when introduced by transformation into the Cm-sensit ive host Streptomyces lividans M252 (Mosher, R. H. Ranade, N. P., Schr empf, H., and Vining, L. C. (1990) J. Gen. Microbiol. 136, 293-301), T ransformants rapidly metabolized Cm to one major product, which was is olated and purified by reversed phase chromatography, The metabolite w as identified by nuclear magnetic resonance spectroscopy and mass spec trometry as 3'-O-phospho-Cm, and was shown to have negligible inhibito ry activity against Cm sensitive Micrococcus luteus, The nucleotide se quence of the S. venezuelae DNA insert in pJV4 contains an open readin g frame (ORF) that encodes a polypeptide (19 kDa) with a consensus mot if at its NH, terminus corresponding to a nucleotide-binding amino aci d sequence (motif A or P-loop; Walker, J. E., Saraste, M., Runswick, M . J., and Gay, N. J. (1982) EMBO J. 1, 945-951). When a recombinant ve ctor containing this ORF as a 1.6-kilobase pair SmaI-SmaI fragment was used to transform S. lividans M252, uniformly Cm-resistant transforma nts were obtained, A strain of S. lividans transformed by a vector in which the ORF had been disrupted by an internal deletion yielded clone s that were unable to phosphorylate Cm, and exhibited normal susceptib ility to the antibiotic. The results implicate the product of the ORF from S. venezuelae as an enzymic effector of Cm resistance in the prod ucing organism by 3'-O-phosphorylation. We suggest the trivial name ch loramphenicol 3'-O-phosphotransferase for the enzyme.