Enzymatic modification of hygromycin B in Streptomyces hygroscopicus.

Antibiotic producing-organisms generally exhibit resistance to the antibiotics that they produce1). Such resistance mechanisms may take the form of target site modification, as is the case with erythromycin2) and thiostrepton3), or enzymatic modification of the antibiotic itself, as has been shown for a number of aminocyclitols4) The existence of these resistance mechanisms in producing organisms is thought to be the means by which the organism avoids growth inhibition by its own antibiotic product although other possibilities cannot be ruled out. For example, in the case of the aminocyclitols the enzymatic modifications may be concerned with specific steps in antibiotic biosynthesis or transport and the fact that the antibiotics are inactivated may be coincidental. It has been suggested that these genes may be evolutionary precursors of the resistance determinants of R-plasmids in clinical isolates of bacteria5). Regardless of the role or evolution of antibiotic modifications in producing organisms, the fact that they are biochemically homologous to the determinants of R-plasmids provides a useful means of predicting possible resistance mechanisms that may be encountered in the therapeutic use of antibiotics. Chemical modifications of antibiotics to block these enzymatic modifications could be undertaken in anticipation of the appearance of resistant bacterial strains. The hygromycin group of antibiotics have not been used extensively, apart from veterinary applications. Hygromycin B, produced by Streptomyces hygroscopicus, has a good range of antibacterial activity; a limited number of R-plasmids have been screened for resistant determinants to this drug but the results were negative (J. D. unpublished observations). Since no resistance mechanism had been detected to date, we have examined extracts of S. hygroscopicus to see if an enzymatic modification exists for hygromycin in the producing organism. Streptomyces hygroscopicus (NRRL 2387) was grown in trypticase soy broth and the cells were harvested, washed and resuspended in buffer. The cells were broken by passing the suspension through a French pressure cell (Aminco) at 1,400 kg/cm2 and the resulting extract centrifuged at 30,000 xg for 30 minutes to remove the cell debris. Samples of the extract were tested for acetyltransferase, adenylyltransferase and phosphotransferase activity, using a variety of substrates. A phosphotransferase activity was detected with hygromycin B and structurally related antibiotics as substrates (Table 1). Note that weak acetyltransferase activity against sisomicin was detected; the enzyme aminoglycoside 3-acetyltransferase appears to be widely distributed in Streptomycetes6). Since previous experience with phosphotransferases had shown that detection was often made difficult because of the presence of potent endogenous ATPase activity, the S. hygroscopicus extract was passed through a column of Bio-Gel P-100; the protein eluting in the void volume was discarded and assay of material retarded by gel filtration chromatography demonstrated potent hygromycin phosphotrans-