Resistance to vernamycin a in streptomyces loidensis.

The vernamycin A (VA)-producing organism, Streptomyces loidensis, is refractory to indigenous drug in vivo. Through the use of an in vitro assay system, it has been shown that the 50S ribosomal subunit (normally the target site of VA) is resistant to drug. To determine whether ribosomal RNA (rRKA) or ribosomal proteins (r proteins) conferred resistance to VA, it became necessary to develop a procedure for reconstituting 50S particles (with the aim of perforning "crossover" experiments with rRNA and r proteins from a VA-sensitive organism). Curiously, 50S particles reconstituted from S, loidensis rRNA and r proteins, were found to exhibit a changed response to VA in vitro (i.e. they were sensitive to drug by comparison with native 50S particles). However, while a rigorous examination of reconstituted 50S particles by a variety of analytical techniques did not reveal an unequivocal reason for the change in response to VA, one novel approach did yield a plausible explanation. Thus, reconstituted 50S particles exposed to VA appeared to lose a ribosomal protein (tentatively identified as homologous with E. coli L7/L12) while no such protein was lost from native 50S particles. Hence, the mechanism of resistance to VA in S. loidensis may involve an increased affinity of the protein for the corresponding ribosome. In support of this notion, the "displacement effect" has been observed (on occasions) with native 50S particles from VA-sensitive organisms. However, it has not been possible to establish unequivocally whether this effect could be the mode of action of the drug. The reconstitution methodology has been adapted and modified to embrace 5OS ribosomal subunits from a variety of Streptomyces spp. and 30S ribosomal subunits from Streptomyces coelicolor. In addition, the methodology has been employed to demonstrate that the resistance determinant for nosiheptide, in the producing organism Streptomyces actuosus, is rRNA.