A circular map of two strains of Streptomyces rimosus was constructed by the analysis of several hundred heteroclones. Segregants from four-point and five-point heteroclones were scored and the markers arranged by the criterion of minimizing the number of quadruple crossovers. Distances between genes were estimated from the analysis of the segregation of pairs of loci, whenever the ratio between the frequency of parental segregants approached the ratio between those of recombinant segregants. The nutritional markers appear to be asymmetrically distributed around the map, the upper arc containing a larger proportion of genes. The arrangement of genes in the upper arc recalls that of s. coelicolor ~3 ( 2 ) , with which s. rimosus forms fertile crosses. The 'empty' regions known in the S. coelicolor map may be absent in S. rimosus.
A general procedure for manipulating protoplasts of three Streptomyces rimosus strains was developed. More than 50% regeneration efficiency was obtained by optimizing the osmotic stabilizer concentrations and modifying the plating procedure. Preparation and regeneration of protoplasts were studied by both phase-contrast and electron microscopy. After cell wall degradation with lysozyme, protoplasts about 1,000 to 1,500 nm in diameter appeared. The reversion process exhibited normal and aberrant regeneration of protoplasts to hyphae and to spherical cells, respectively. Spherical cells contained no ot,E-LL-diaminopimelic acid and were colorless or red after Gram staining. They showed consistent stability during at least five subsequent subcultivations. However, the omission of glycine from the precultivation medium reduced the unusual process of regeneration almost completely. After normal protoplast regeneration, the production of oxytetracycline by single isolates was not affected.
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