Two alleles for viomycin-capreomycin resistance (vic) in Mycobacterium smegmatis affect ribosome structures. One (vicA) affects a component of 508 subunits and the other (vicB) affects a component of 30S subunits. The locus for neomycin-kanamycin resistance (nek), which is linked to vicA and vicB, affects a component of 30s subunits. Although the erythromycin resistance locus (ery) is linked to vic and nek, no ribosomal alterations could be detected. Mutations at the streptomycin locus (str) not linked to vic and nek caused alterations of 30S subunits.In our previous paper (34), we reported that viomycin (VM)-resistant mutants isolated from Mycobacterium smegmatis ATCC 14468 had altered ribosomes. One of these mutants had altered 50S subunits. By contrast, others had altered 30S subunits. Thus, it was concluded that VM action could be counteracted to some extent by changes in both 50S and 30S subunits. These altered ribosomes demonstrated pleiotropic resistance to kanamycin (KM) and other antibiotics (33). The genetics of these mutants have not been reported.Recent studies reveal that there is genetic recombination between two different strains of M. smegmatis (24, 31). Using compatible strains of M. smegmatis Rabinowitchi and PM5, we found close linkage between the loci conferring resistance to neomycin-kanamycin (nek), viomycin-capreomycin (vic), and erythromycin (ery). These three genes were closely linked to the genes for arginine biosynthesis (argA, argB). No linkage was observed between str and nek or vic. By the complementation tests, we also noticed that vic resistance was determined by mutations in two cistrons (23).As a continuation of this work, the localization of drug resistance on ribosomal subunits of these genetically characterized mutants has been tested. Based on the results obtained in this study and observations ofthe previous publications (23,28,33,34), genetic and biochemical aspects of drug resistance of M. smegmatis will be discussed.

Genetic analyses of the loci conferring resistance to antibiotics known to affect protein synthesis were made employing the conjugation system of Mycobacterium smegmatis. By the complementation tests, vic (viomycin-capreomycin-resistant) mutants were classified into two different groups; cistrons A and B. Neomycin-kanamycin-resistant (nek) mutants, on the contrary, fall into a single cistron. Erythromycin-resistance (ery) locus was linked to vic-nek region. The map order deduced was argB, argA, (vicA, vicB), nek, ery. Biochemical studies showed that strains resistant to streptomycin, neomycinkanamycin or viomycin-capreomycin had altered ribosomes.In the previous report, we analyzed the linkage relationships among the markers for resistance to streptomycin (str), neomycinkanamycin (nek), and viomycin-capreomycin (vic) in Mycobacterium smegmatis [4].Since these markers were presumed to define genes for ribosomal components, and known to form a cluster in a region of chromosome in Escherichia coli [1,2] and Bacillus subtilis [3], linkage relationships among these loci in mycobacteria were of particular interest from the phylogenetical point of view. The results showed that nek and vic loci were closely linked with each other and also linked to the genes for the arginine biosynthesis (argA and argB). No linkage was observed, however, between str and nek or vic. We also showed that in certain recombinant types, nek and vic alleles formed stable heterogenotes. Accordingly, it is expected that complementation tests are possible between the different vic or nek mutations, because vic" or nekr is recessive to vics or neks alleles [4].

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Viomycin-resistant strains isolated from Mycobacterium smegmatis demonstrated pleiotropic resistance to tuberactinomycin-N, capreomycin, streptomycin, and kanamycin as a result of mutational alteration of ribosomes, even though they were selected for resistance to a single antibiotic. The pleiotropic drug resistance of three mutants isolated by stepwise selection for resistance to viomycin was due to alteration of the 30S ribosomal subunit. One mutant, strain A, isolated independently by multiple-step selection to viomycin resistance, was resistant to viomycin, tuberactinomycin-N, and capreomycin through an alteration of the 50S ribosomal subunit, whereas it was sensitive to kanamycin but resistant to streptomycin through an alteration of the 30S ribosomal subunit. Three streptomycin-resistant strains, which were isolated by one-step selection at a high concentration of streptomycin, did not show significant co-resistance to any other antibiotics tested in culture and cell-free systems; streptomycin resistance in these mutants was localized on the 30S ribosomal subunit.Pleiotropic drug resistance associated with viomycin (VM) resistance in mycobacteria has been reported (6,9,12,13,14,15,25,26,31,32,33,34,37,38,39). Three possible mechanisms for this drug resistance might be considered: (i) enzymatic inactivation of the drugs (17), (ii) alteration of the membrane leading to a change in permeability, and (iii) alterations at the site of action of the antibiotics, e.g., ribosomes (44).As a continuation of our work, pleiotropic drug resistance was studied in culture and in cell-free systems. The results indicate that mutational alteration of the VM binding site on ribosomes led to pleiotropic drug resistance in Mycobacterium smegmatis.The results were presented in abstract form at the XXIInd International Tuberculosis Conference (T. Yamada et al., XXIInd Int. Tuberc.

Viomycin-resistant strains were isolated from Mycobacterium smegmatis . Ribosomes were isolated and tested for drug resistance in subcellular systems containing poly(U) as messenger ribonucleic acid. Resistance to viomycin in these strains was due to altered ribosomes. Further analysis showed that viomycin resistance of two mutants with low level resistance (20 μg/ml) was due to altered 30 S ribosomal subunits. Another mutant that was highly resistant to viomycin (1 mg/ml), however, had altered 50 S ribosomal subunits.