Isolation of a protoplast-type L-form fromStreptomyces hygroscopicus

1982

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(Eingegangen a m 18.12.1981) L-form colonies from S. hygroscopicus, S. griseus and 8. levoris were isolated after incubation of lysozyme protoplasts on an osmotically stabilized complex agar medium. Unstable and stable L-forms grow on solid and in liquid media. 1,-form colonies are 5-10 times smaller than normal colonies and show a typical morphology for each species. I n ultrathin sections L-form cells are characterized by nucleoid areas with typical core-like structures, by a ribosome-rich cytoplasm with different inclusion bodies, and by a cytoplasmic membrane. Because there are no cell wall structures L-forms of the three Streptomyces species belong to the protoplast type.Analysis of cell size and cell shape shows a variation in diameter and a cell propagation by regular and irregular division-and budding-like processes. Many L-form cells contain more than two chromosomes.The results are discussed with regard to the cellular organisation of streptomycetes and the nature of the stable L-form.Although much work has been done about protoplasts of streptomycetes (DOUGLAS et al. 1958, HOPWOOD et al. 1977, KOCHKINA and RAUTENSTEIN 1968, OKANISHI et al. 1966, 1974, SAGARA et al. 1971 no data exist about Streptomyces L-forms, i.e. about growing and dividing protoplasts or spheroplasts of these organisms.Cells of streptomycetes growing and propagating in the protoplast state as L-forms might be of great interest for several reasons: they offer the possibility of obtaining Streptomyces cells more uniform in age and physiological state, from which cellular components can be isolated more easily; they seem to be useful objects to study physiological and biochemical questions, especially the biosynthesis of primary and secondary metabolites; and they are good candidates for genetic experiments, particularly for fusion, tranformation and transfection processes.I n two papers (BAUDLER and GUMPERT 1979, GUMPERT 1978) we have described the isolation of Streptomyces hygroscopicus L-form. I n this report colony morphology, growth characteristics, and ultrastructure of L-forms from three Streptomyces species are summarized. Spores conserved in earth (LUVOS Heilerde) were used for maintaining the strains. Induction of L-form growth: Streptomyces strains were grown for 16-20 h a t 28 "C in a complex medium composed of mineral salts, glucose (l%), peptone (0.5y0), yeast extract (0.4%), and glycine (lye). Mycelia arc transformod into protoplasts by lysozyme treatment (2000 yg/ml) in protoplasting medium according to OKANISHI et al. (1974). Materials and methods

Growth characteristics and ultrastructure of protoplast type L‐forms from Streptomycetes

1982

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Bioconversions of a macrolide glycoside by growing L‐form cells of Streptomyces hygroscopicus

Gräfe

1983

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Growing L-form cells of Streptomyces hygroscopicus were shown to carry out 3-0-acylation and 14-C-hydroxylation of a macrolide glycoside suggesting that both types of bioconversion do neither require the intact cell wall nor the periplasmic space.Protoplasts of streptomycetes can be used as a tool for studies on the biosynthesis of antibiotics and other secondary metabolites ( KELLER and KLEINKAUF 1977, HITCHCOCK andKATZ 1978). Because they lack the cell wall, metabolic conversions will be supported which do not require the cell wall and the periplasmic space. I n contrast to protoplasts, which represent resting cells, the so-called L-forms of streptomycetes (BAUDLER and GUMPERT 1979, GUMPERT 1980) also lack a cell wall but are growing and propagating cells that can be used in biosynthetic studies. But up to now, no information has been available about their capacity to carry out particular bioconversions of secondary metabolites. With regard to the biosynthesis of peptide antibiotics performed by soluble cytoplasmic enzymes, the cellular localization of macrolide-producing enzymes is rather uncertain. There is evidence, however, that the intermediates of macrolide formation administered to the medium can undergo specific conversions that may be a consequence of their penetration inside the cells (KITAO et aZ. 1979a, b, c). On the other hand, at least in the biogenesis of the polyene macrolides, glycosidations appear to occur a t the cytoplasmic membrane or in the periplasmic space during the transport of the aglycones into the medium ( MARTIN 1977, MARTIN andGIL 1979). Experiments with protoplast sof macrolide-producing streptomycetes in which any antibiotic-generating machinery must necessarily be localized a t the cytoplasmic membrane or in the cytosole have not so far been published. Therefore, in the present work we checked the capacity of growing L-form cells of Streptomyces hygroscopicus both to acetylate a macrolide glycoside at the C-3-OH position andtooxidizethe aglycone a t (2-14. We sought to answer the question: is 3-0-acylation, as one of the final steps in the biogenesis of 16-membered macrolide antibiotics ( KITAO et al. 1979a, b, c), dependent on an intact periplasmic space and cell wall structure. Materials and methodsOrganisms and medium: The L-form was isolated from 8. hygroscopicus IMET J A 6599-NG33-354r (identicll with ZIMET 40668) obtained from the strain collection of the Central Institute of Microbiology and Experimental Therapy, Jena. Formation of lysozyme protoplasts, induction of L-form growth and cultivation of the L-form strain were described in a previous paper (BAUDLER and GIJMPERT 1979).

Ultrastructural characterization of core structures and paracrystalline inclusion bodies in L‐form cells of streptomycetes

1983

Protoplast type L‐form cells of Streptomyces hygroscopicus and S. griseus contain different types of inclusion bodies. Cytoplasmic cores and paracrystalline structures are peculiar inclusions which could not be observed in normal parent bacteria. The cytoplasmic cores are 1–4 μm long and 0.05–0.25 μm broad straight and stiff non‐tubular structures consisting of homogeneous moderate electron opaque material. Paracrystalline inclusions have side‐lengths between 0.2 and 0.5 μm and show a characteristic pattern of 15–20 nm thick straight dark lines and electron lucent intervening spaces of 20–30 nm. Both cytoplasmic cores and paracrystalline inclusions are apparently proteins. Their occurrence in L‐form cells indicates an altered synthesis of one or several proteins in these cell types.